Natural Benzo/Acetophenones as Leads for New Synthetic Acetophenone Hybrids Containing a 1,2,3-Triazole Ring as Potential Antifouling Agents

Marine biofouling is a natural process that represents major economic, environmental, and health concerns. Some booster biocides have been used in biofouling control, however, they were found to accumulate in environmental compartments, showing negative effects on marine organisms. Therefore, it is urgent to develop new eco-friendly alternatives. Phenyl ketones, such as benzophenones and acetophenones, have been described as modulators of several biological activities, including antifouling activity (AF). In this work, acetophenones were combined with other chemical substrates through a 1,2,3-triazole ring, a strategy commonly used in Medicinal Chemistry. In our approach, a library of 14 new acetophenone–triazole hybrids was obtained through the copper(I)-catalyzed alkyne-azide cycloaddition “click” reaction. All of the synthesized compounds were evaluated against the settlement of a representative macrofouling species, Mytilus galloprovincialis, as well as on biofilm-forming marine microorganisms, including bacteria and fungi. The growth of the microalgae Navicula sp. was also evaluated after exposure to the most promising compounds. While compounds 6a, 7a, and 9a caused significant inhibition of the settlement of mussel larvae, compounds 3b, 4b, and 7b were able to inhibit Roseobacter litoralis bacterial biofilm growth. Interestingly, acetophenone 7a displayed activity against both mussel larvae and the microalgae Navicula sp., suggesting a complementary action of this compound against macro- and microfouling species. The most potent compounds (6a, 7a, and 9a) also showed to be less toxic to the non-target species Artemia salina than the biocide Econea®. Regarding both AF potency and ecotoxicity activity evaluation, acetophenones 7a and 9a were put forward in this work as promising eco-friendly AF agents.


Introduction
Biofouling is an inevitable natural phenomenon that occurs continually on marine vessels and other submerged structures. Propulsive fuel increase, required to overcome the increased drag created by fouled hulls, constitutes significant costs for the maritime industry, and there is a substantial industrial and commercial interest in controlling the biofouling process, namely by dry-docking, scraping, and re-painting hulls [1].
The natural process of marine biofouling starts with the deposition of organic material that encourages the attachment of marine bacteria, diatoms, and fungi, and the colonization of macroorganisms, such as macroalgae, sponges, cnidarians, polychaetes, mollusks, barnacles, bryozoans, and tunicates [2]. Therefore, it is important to target diverse levels of fouling when trying to efficiently combat biofouling.
Antifouling (AF) coatings, used to prevent biofouling, are of great environmental concern. Biocides, after being released from the AF coating, can accumulate in the environment, predominantly in areas where there is intense boating activity. Most biocides are persistent and do not undergo degradation rapidly, bioaccumulating through the food chain and showing negative effects in marine organisms. The deposition in sediments is also a concern, as they will be continually released into the water. Therefore, there is a renewed effort to develop harmless alternatives. In addition to the search of new eco-friendly AF agents, other technologies to prevent marine biofouling have been developed, such as silicone polymer AF coatings, biomimetic AF coatings, or photocatalytic AF coatings; however, these technologies are hard to apply. Therefore, the development of AF compounds with low environmental impact remains one of the most useful strategies in the development of new effective AF coatings [3].
In recent years, marine phenyl ketones, such as benzophenones A-D, have been reported in the literature as antibacterial and antifungal agents ( Figure 1) [4][5][6]. Moreover, the benzophenone scaffold has been used for the preparation of some AF coatings (E, Figure 1) [7]. Molecular simplification of the benzophenone structure has also been applied in the development of promising AF agents: 2,4-dihydroxyacetophenone (F, Figure 1) was shown to significantly inhibit the spore attachment of a green fouling alga (Ulva pertusa) and, after being incorporated in a controlled depletion paint, a significant decrease in fouling biomass was observed [8]. Therefore, phenyl ketones, such as benzophenones and acetophenones, show potential as new leads to develop eco-friendly and sustainable AF agents for the marine industry.
Mar. Drugs 2021, 19,682 2 of 17 the increased drag created by fouled hulls, constitutes significant costs for the maritime industry, and there is a substantial industrial and commercial interest in controlling the biofouling process, namely by dry-docking, scraping, and re-painting hulls [1]. The natural process of marine biofouling starts with the deposition of organic material that encourages the attachment of marine bacteria, diatoms, and fungi, and the colonization of macroorganisms, such as macroalgae, sponges, cnidarians, polychaetes, mollusks, barnacles, bryozoans, and tunicates [2]. Therefore, it is important to target diverse levels of fouling when trying to efficiently combat biofouling.
Antifouling (AF) coatings, used to prevent biofouling, are of great environmental concern. Biocides, after being released from the AF coating, can accumulate in the environment, predominantly in areas where there is intense boating activity. Most biocides are persistent and do not undergo degradation rapidly, bioaccumulating through the food chain and showing negative effects in marine organisms. The deposition in sediments is also a concern, as they will be continually released into the water. Therefore, there is a renewed effort to develop harmless alternatives. In addition to the search of new ecofriendly AF agents, other technologies to prevent marine biofouling have been developed, such as silicone polymer AF coatings, biomimetic AF coatings, or photocatalytic AF coatings; however, these technologies are hard to apply. Therefore, the development of AF compounds with low environmental impact remains one of the most useful strategies in the development of new effective AF coatings [3].
In recent years, marine phenyl ketones, such as benzophenones A-D, have been reported in the literature as antibacterial and antifungal agents ( Figure 1) [4][5][6]. Moreover, the benzophenone scaffold has been used for the preparation of some AF coatings (E, Figure 1) [7]. Molecular simplification of the benzophenone structure has also been applied in the development of promising AF agents: 2,4-dihydroxyacetophenone (F, Figure 1) was shown to significantly inhibit the spore attachment of a green fouling alga (Ulva pertusa) and, after being incorporated in a controlled depletion paint, a significant decrease in fouling biomass was observed [8]. Therefore, phenyl ketones, such as benzophenones and acetophenones, show potential as new leads to develop eco-friendly and sustainable AF agents for the marine industry. In this work, several acetophenones were synthesized, and the AF activity was assessed against the settlement of Mytilus galloprovincialis, a heavy macrofouler, five strains of biofilm-forming marine bacteria (Cobetia marina, Vibrio harveyi, Pseudoalteromonas atlantica, Halomonas aquamarina, and Roseobacter litoralis), a marine diatom (Navicula sp.), and three fungal strains (Candida albicans, Aspergillus fumigatus, and Trichophyton rubrum). The In this work, several acetophenones were synthesized, and the AF activity was assessed against the settlement of Mytilus galloprovincialis, a heavy macrofouler, five strains of biofilm-forming marine bacteria (Cobetia marina, Vibrio harveyi, Pseudoalteromonas atlantica, Halomonas aquamarina, and Roseobacter litoralis), a marine diatom (Navicula sp.), and three fungal strains (Candida albicans, Aspergillus fumigatus, and Trichophyton rubrum). The ecotoxicity and bioaccumulative potential of the most promising compounds was also evaluated in the discovery of eco-friendly compounds.

Synthesis and Structure Elucidation
Combining different bioactive ligands/pharmacophores into a single molecule is a strategy currently employed in Medicinal Chemistry even for AF activity [9,10]. Triazole derivatives have undeniable importance in Medicinal Chemistry, displaying several bioactivities, such as antimicrobial, which includes the family of the so-called "azoles" used in the treatment of fungal infections [11]. The incorporation of a 1,2,3-triazole ring in the benzophenone and acetophenone scaffolds was used to generate compounds with antimicrobial activity [12,13]. Moreover, the presence of triazoles was already associated to AF activity of biocides [14]. For instance, bromotyramine hybrids containing a 1,2,3-triazole ring were described as inhibitors of biofilm formed by marine bacteria [15]. Recently, previous research developed by our group has identified a chalcone glycoside with a 1,2,3-triazole moiety with promising AF activity against macro and microfouling species, without ecotoxicity to a non-target marine organism [16].
Based on these data, and considering the importance of both 1,2,3-triazole and benzo/acetophenone moieties, a series of 14 new 1,2,3-triazole tethered acetophenones (3a-9b) was synthesized ( Figure 2). ecotoxicity and bioaccumulative potential of the most promising compounds was also evaluated in the discovery of eco-friendly compounds.

Synthesis and Structure Elucidation
Combining different bioactive ligands/pharmacophores into a single molecule is a strategy currently employed in Medicinal Chemistry even for AF activity [9,10]. Triazole derivatives have undeniable importance in Medicinal Chemistry, displaying several bioactivities, such as antimicrobial, which includes the family of the so-called "azoles" used in the treatment of fungal infections [11]. The incorporation of a 1,2,3-triazole ring in the benzophenone and acetophenone scaffolds was used to generate compounds with antimicrobial activity [12,13]. Moreover, the presence of triazoles was already associated to AF activity of biocides [14]. For instance, bromotyramine hybrids containing a 1,2,3-triazole ring were described as inhibitors of biofilm formed by marine bacteria [15]. Recently, previous research developed by our group has identified a chalcone glycoside with a 1,2,3triazole moiety with promising AF activity against macro and microfouling species, without ecotoxicity to a non-target marine organism [16].
The incorporation of the triazole was accomplished by the CuAAC reaction (Table 1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
The incorporation of the triazole was accomplished by the CuAAC reaction (Table  1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields (Table 1). The incorporation of the triazole was accomplished by the CuAAC reaction (Table  1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields ( Table 1). The incorporation of the triazole was accomplished by the CuAAC reaction (Table  1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields ( Table 1). The incorporation of the triazole was accomplished by the CuAAC reaction (Table  1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields ( Table 1). The incorporation of the triazole was accomplished by the CuAAC reaction (Table  1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields ( Table 1). The incorporation of the triazole was accomplished by the CuAAC reaction (Table  1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields ( Table 1). The incorporation of the triazole was accomplished by the CuAAC reaction (Table  1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields (Table 1). The incorporation of the triazole was accomplished by the CuAAC reaction (Table  1). To obtain structure-activity relationship (SAR) information, several structure-related acetophenones with diversified substitution patterns were explored: aromatic nitriles (3a and 3b), since isocyanides, their isomers, were extensively studied in AF activity [19]; aromatic halogens to combine with acetophenones (4a-6b) and obtain 1,4 disubstituted 1,2,3-triazoles as the literature reports hybrids with these substitutions as having antimicrobial activity [20][21][22]; and the other two substituents, namely an aliphatic alcohol (7a and 7b) and a glucose acetamide (9a and 9b) were also included for SAR studies.
Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields (Table 1). The newly synthesized compounds 3a-9b were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) (Supplementary material, Figure S1-S28). Compounds 2a and 2b were already Acetophenones 2a and 2b reacted with the selected azides in the presence of catalytically copper (I) ions formed by the reduction of copper (II) from CuSO 4 by sodium ascorbate, to afford derivatives 3a-9b in 30-84% yields ( Table 1).
The newly synthesized compounds 3a-9b were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) (Supplementary material, Figure S1-S28). Compounds 2a and 2b were already described in the literature and NMR data are in accordance with the data already published [23,24].
The NMR spectra of compounds 3a-9b showed the characteristic signals of the acetophenone scaffold. The signals corresponding to the triazole ring were also observed (δ H -triazole: 7.71-9.00 s, δ C -H triazole: 134.7-145.8 and 118.2-121.2). Regarding compounds 3a-6b, 8a, and 8b, signals corresponding to the aromatic protons were observed. Additionally, a carbon signal corresponding to C≡N (δ C 118.2 and 118.6, respectively) was observed for compounds 3a and 3b. The NMR spectra of compounds 9a and 9b showed several proton and carbon signals corresponding to the acetamide glucose moiety. Aliphatic protons and carbons were observed in the NMR spectra of compounds 7a and 7b, and proton and carbons signals assigned to the methoxy group of compounds 8a and 8b were observed in the NMR spectra of these compounds.

Mussel Larvae Anti-Settlement Activity
The presence of macrofouling species has a dominant influence on ship drag [25], increasing fuel consumption. The larvae of some species of bryozoans, polychaetes, mollusks, and some other biofoulers may adhere even before biofilm formation. Mussel plantigrade larvae are highly specialized in adhesion to the submerged surfaces and the fixation is made through the production of byssal threads. Therefore, mussel plantigrade larvae were selected to assess the AF activity of the newly synthesized 1,2,3-triazolylacetophenones (3a-9b). The ability of the synthesized compounds to inhibit the settlement of Mytilus galloprovincialis larvae was first screened at 50 µM ( Figure 3).

9b
OH H 35 The newly synthesized compounds 3a-9b were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) (Supplementary material, Figure S1-S28). Compounds 2a and 2b were already described in the literature and NMR data are in accordance with the data already published [23,24].
The NMR spectra of compounds 3a-9b showed the characteristic signals of the acetophenone scaffold. The signals corresponding to the triazole ring were also observed (δHtriazole: 7.71-9.00 s, δC-H triazole: 134.7-145.8 and 118.2-121.2). Regarding compounds 3a-6b, 8a, and 8b, signals corresponding to the aromatic protons were observed. Additionally, a carbon signal corresponding to C≡N (δC 118.2 and 118.6, respectively) was observed for compounds 3a and 3b. The NMR spectra of compounds 9a and 9b showed several proton and carbon signals corresponding to the acetamide glucose moiety. Aliphatic protons and carbons were observed in the NMR spectra of compounds 7a and 7b, and proton and carbons signals assigned to the methoxy group of compounds 8a and 8b were observed in the NMR spectra of these compounds.

Mussel Larvae Anti-Settlement Activity
The presence of macrofouling species has a dominant influence on ship drag [25], increasing fuel consumption. The larvae of some species of bryozoans, polychaetes, mollusks, and some other biofoulers may adhere even before biofilm formation. Mussel plantigrade larvae are highly specialized in adhesion to the submerged surfaces and the fixation is made through the production of byssal threads. Therefore, mussel plantigrade larvae were selected to assess the AF activity of the newly synthesized 1,2,3-triazolylacetophenones (3a-9b). The ability of the synthesized compounds to inhibit the settlement of Mytilus galloprovincialis larvae was first screened at 50 μM ( Figure 3). Among the 14 tested compounds, 6a, 7a, and 9a presented a percentage of settlement ≤ 40% and were selected as positive hits for dose-response studies to determine the EC50 and LC50/EC50 values regarding anti-settlement activity ( Table 2). In general, when comparing the results of compounds with the same substitution on the 1,2,3-triazole ring, it was found that the presence of two methoxy groups at C-3′ and C-5′ on the phenyl ketone Among the 14 tested compounds, 6a, 7a, and 9a presented a percentage of settlement ≤ 40% and were selected as positive hits for dose-response studies to determine the EC 50 and LC 50 /EC 50 values regarding anti-settlement activity ( Table 2). In general, when comparing the results of compounds with the same substitution on the 1,2,3-triazole ring, it was found that the presence of two methoxy groups at C-3 and C-5 on the phenyl ketone core is more beneficial for the mussel larvae anti-settlement activity than the presence of hydroxyl groups at C-2 . The three compounds presented an EC 50 < 25 µg·mL −1 , a value recommended by the U.S. Navy program for anti-foulants [26]. Acetophenone derivative 9a (EC 50 = 9.94 µg·mL −1 ), containing an acetamide glucose moiety was the most effective larval settlement inhibitor, followed by compounds 6a (EC 50 = 11.20 µg·mL −1 ) with an aromatic chlorine, and 7a (EC 50 = 13.46 µg·mL −1 ) with an aliphatic alcohol. Considering toxicity, none of these compounds caused mortality to the target species M. galloprovincialis plantigrades to the highest concentration tested (200 µM).

Biofilm-Forming Marine Microorganism Growth Inhibitory Activity
The settlement of some macroorganisms might be enhanced by the presence of microbial biofilms present in submerged surfaces [27]. Microbial biofilms composed of bacteria, fungi, diatoms, unicellular algae, and protozoa, represent an important component of fouling communities. Therefore, synthesized acetophenones were also evaluated for their ability to inhibit the growth of marine biofilm-forming bacteria (Vibrio harveyi, Cobetia marina, Halomonas aquamarina, Pseudoalteromonas atlantica, and Roseobacter litoralis), fungi (Candida albicans, Aspergillus fumigatus, and Trichophyton rubrum), and microalgae (Navicula sp.). Results showed that compounds 3b, 4b, and 7b were able to significantly inhibit R. litoralis biofilm growth ( Figure 4A). In contrast to the mussel larvae anti-settlement activity, the presence of hydroxyl groups at C-2 on the phenyl ketone core seems to be more beneficial for the antibacterial activity.  Figure 4B). EC 50 was not possible to estimate for compound 7b, given the low activity at the higher concentrations tested.
Fungi are also important players in the marine biofouling colonization cascade, as they are also able to produce biofilms. Therefore, to evaluate the AF potential of compounds 3a-9b against microfouling species, their antifungal activity was evaluated against three biofilm-forming fungi species, Candida albicans [28], Aspergillus fumigatus [29], and Trichophyton rubrum [30], by determining minimum inhibitory concentration (MIC). None of the compounds tested showed activity against the fungal strains, with MICs higher than the maximum tested concentration (128 µg·mL −1 ).
Marine diatoms colonize very quickly and effectively submerged surfaces by secreting adhesive extracellular polymer substances, and thus are a good representative of fouling microalgae. The ability to inhibit the growth of the biofilm-forming marine diatom Navicula sp. was also evaluated but only for compounds with the most significant AF activity (6a, 7a, and 9a). From the three tested compounds, acetophenone 7a showed inhibitory activity against Navicula sp. growth ( Figure 5  Fungi are also important players in the marine biofouling colonization cascade they are also able to produce biofilms. Therefore, to evaluate the AF potential of co pounds 3a-9b against microfouling species, their antifungal activity was evalua against three biofilm-forming fungi species, Candida albicans [28], Aspergillus fumiga [29], and Trichophyton rubrum [30], by determining minimum inhibitory concentrat (MIC). None of the compounds tested showed activity against the fungal strains, w MICs higher than the maximum tested concentration (128 µ g·mL −1 ).
Marine diatoms colonize very quickly and effectively submerged surfaces by sec ing adhesive extracellular polymer substances, and thus are a good representative of fo ing microalgae. The ability to inhibit the growth of the biofilm-forming marine diat Navicula sp. was also evaluated but only for compounds with the most significant AF tivity (6a, 7a, and 9a). From the three tested compounds, acetophenone 7a showed inh itory activity against Navicula sp. growth ( Figure 5

Artemia Salina Ecotoxicity Bioassay
The most active compounds (6a, 7a, and 9a,) were further submitted to ec assays against non-target organisms. The standard ecotoxicity bioassay using shrimp Artemia salina was used due to its easy culture, short generation time, co tan distribution, and commercial availability of their eggs in the latent form [3 tested compounds were found to be less toxic to Artemia salina at both conce tested (25 and 50 μM) than the commercial biocide ECONEA ® (100% lethality) [3 acetophenones 7a and 9a mortality rates not significantly different from negativ ( Figure 6).

Artemia Salina Ecotoxicity Bioassay
The most active compounds (6a, 7a, and 9a,) were further submitted to ecotoxicity assays against non-target organisms. The standard ecotoxicity bioassay using the brine shrimp Artemia salina was used due to its easy culture, short generation time, cosmopolitan distribution, and commercial availability of their eggs in the latent form [31,32]. All tested compounds were found to be less toxic to Artemia salina at both concentrations tested (25 and 50 µM) than the commercial biocide ECONEA ® (100% lethality) [33], being acetophenones 7a and 9a mortality rates not significantly different from negative control ( Figure 6). ECONEA ® was added for comparative purposes [33]. * Indicates significant differences against C-(Dunnett test, p  0.01).

In Silico Evaluation of Bioaccumulation Potential
One of the major concerns on new AF agents is the potential bioaccumulation in marine organisms. Compounds are considered potential bioaccumulative if the LogKow (octanol-water partition coefficient) is higher or equal to 3. Therefore, in silico prediction of LogKow values was calculated for the most promising compounds 7a and 9a. Both compounds presented a LogKow value lower than 3 (0.38 for 7a and −2.32 for 9a), suggesting their low potential for bioaccumulation [34].

General Methods
Reactions were monitored by analytical thin-layer chromatography (TLC). Purifications of compounds were carried out by flash column chromatography using Macherey-Nagel silica gel 60 (0.04-0.063 mm) and crystallization. Melting points were obtained in a Köfler microscope and are uncorrected. 1 H and 13 C NMR spectra were taken in CDCl3 or DMSO-d6 at room temperature, on Bruker Avance 300 instruments (300.13 MHz for 1 H and 75.47 MHz for 13 C). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference; 13 C NMR assignments were made by 2D (HSQC and HMBC) NMR experiments (long-range 13   ECONEA ® was added for comparative purposes [33]. * Indicates significant differences against C-(Dunnett test, p < 0.01).

In Silico Evaluation of Bioaccumulation Potential
One of the major concerns on new AF agents is the potential bioaccumulation in marine organisms. Compounds are considered potential bioaccumulative if the LogKow (octanolwater partition coefficient) is higher or equal to 3. Therefore, in silico prediction of LogKow values was calculated for the most promising compounds 7a and 9a. Both compounds presented a LogKow value lower than 3 (0.38 for 7a and −2.32 for 9a), suggesting their low potential for bioaccumulation [34].

General Methods
Reactions were monitored by analytical thin-layer chromatography (TLC). Purifications of compounds were carried out by flash column chromatography using Macherey-Nagel silica gel 60 (0.04-0.063 mm) and crystallization. Melting points were obtained in a Köfler microscope and are uncorrected. 1 H and 13 C NMR spectra were taken in CDCl 3 or DMSO-d 6 at room temperature, on Bruker Avance 300 instruments (300.13 MHz for 1 H and 75.47 MHz for 13 C). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference; 13 C NMR assignments were made by 2D (HSQC and HMBC) NMR experiments (long-range 13

Synthesis of Propargyloxy Acetophenone 2a and 2b
Compounds 2a and 2b were synthesized and characterized following previously reported methods and 1 H and 13 C NMR data were in accordance with the previously reported [24] and [16,23], respectively.

Mussel (Mytilus galloprovincialis) Larvae Anti-Settlement Activity
Mussel (Mytilus galloprovincialis) plantigrades were collected in juvenile aggregates during low neap tides at Memória beach, Matosinhos, Portugal (41 • 13 59 N; 8 • 43 28 W). In the laboratory, mussel plantigrade larvae (0.5-2 mm) were isolated in a binocular magnifier (Olympus SZX2-ILLT, Tokyo, Japan) to a petri dish with filtered seawater, and those with functional foot and competent exploring behavior were selected to the bioassays. Compounds were screened at 50 µM in 24-well microplates with 4-well replicates per condition and 5 larvae per well, for 15 h, in the darkness at 18 ± 1 • C, according to the previously reported [35,36]. All compounds that caused more than 60% of settlement inhibition (≤40% of settlement) in the screening bioassay were considered active and selected for the determination of the semi-maximum response concentration that inhibited 50% of the larval settlement (EC 50 ), at compounds concentrations of 3.125, 6.25, 12.5, 25, 50, 100, and 200 µM.

Biofilm-Forming Marine Bacteria Growth Inhibitory Activity
Five strains of marine biofilm-forming bacteria from the Spanish Type Culture Collection (CECT): Cobetia marina CECT 4278, Vibrio harveyi CECT 525, Halomonas aquamarina CECT 5000, Pseudoalteromonas atlantica CECT 570, and Roseobacter litoralis CECT 5395 were selected for antibacterial screening. The experimental procedure was performed according to the previously reported [35,36]. Briefly, bacteria were inoculated and incubated for 24 h at 26 • C in marine broth (Difco) at an initial density of 0.1 (OD600) in 96-well flat-bottom microtiter plates and exposed to the test compounds at 15 µM. Bacterial growth inhibition in the presence of the compounds was determined in quadruplicate at 600 nm using a microplate reader (BioTek Synergy HT, Winooski, VT, USA). Negative and positive controls used were a solution of marine broth with 0.1% DMSO, and a solution of marine broth with penicillin-streptomycin-neomycin, respectively. Bacterial growth inhibition calculations were made based in the formula ((Mc-Mt)/Mc) × 100, where Mc is the mean of the four replicates from negative control, and Mt is the mean of four replicates from each of the tested compounds.

Antifungal Susceptibility Testing
The antifungal activity was evaluated against Candida albicans ATCC 10231, Aspergillus fumigatus ATCC 204305, and Trichophyton rubrum-FF5. Candida krusei ATCC 6258 was used for quality control. To guarantee the purity and viability, the strains were sub-cultured before each assay on Sabouraud dextrose agar (BioMérieux, Marcy l'Etoile, France). RPMI-1640 broth medium pH 7.0, with L-glutamine and without bicarbonate (Biochrom) and buffered with 0.165 mol·L −1 of 3-(N-morpholino)-propanesulfonic acid (MOPS, Sigma-Aldrich, St. Louis, MO, USA), was used on the evaluation of the antifungal activity. The MICs were evaluated using the broth microdilution method and in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI) reference documents: M27-A3 for yeasts [37] and M38-A2 [38] for filamentous fungi. Two-fold serial dilutions of compounds were prepared within the concentration range of 8-128 µg·mL −1 . Yeast cells suspensions were prepared to obtain an inoculum of 1-5 × 10 3 CFU·mL −1 . For filamentous fungi, a spore suspension is prepared, and cell density was adjusted to obtain the adequate inoculum (for dermatophytes 1-3 × 10 3 CFU·mL −1 and 0.4-5 × 10 4 CFU·mL −1 for Aspergillus fumigatus). Equal volumes of compound dilution in RPMI and cell suspension in RPMI were added in the wells of the microplate. Controls performed were sterility control, growth control, and quality control. Quality control was performed with an ATCC reference strain (Candida krusei ATCC 6258) with a commercial antifungal compound, voriconazole (Pfizer) ranging between 0.25-1 µg·mL −1 . The plates were incubated aerobically at 35 • C for 48 h for Candida albicans and Aspergillus fumigatus and at 25 • C for 5-7 days for dermatophytes. MICs were determined as the lowest concentrations resulting in 100% growth inhibition, in comparison to the compound-free controls. All the compounds were tested independently three times.

Biofilm-Forming Marine Diatoms Growth Inhibitory Activity
The anti-microalgal activity of the most promising compounds was also evaluated against a benthic marine diatom, Navicula sp., purchased from the Spanish Collection of Algae (BEA), according to the previously reported [16]. Briefly, diatom cells were inoculated in f/2 medium (Sigma) at an initial concentration of 2-4 × 10 6 cells·mL −1 and grown in 96-well flat-bottom microtiter plates for 14 days in continuous light at 20 • C. Navicula sp. growth inhibition in the presence of each compound at 25 µM was determined in quadruplicate, and cells were counted using a Neubauer counting chamber. Growth inhibition was calculated based in the formula ((Mc-Mt)/Mc) × 100, where Mc is the mean of the cell counts of the four replicates from negative control, and Mt is the mean of the cell counts of the four replicates from each of the tested compounds. Positive control with cycloheximide (3.55 µM) and negative control with f/2 medium 0.1% DMSO were included.

Artemia Salina Ecotoxicity Bioassay
The brine shrimp (Artemia salina) nauplii lethality test was used to determine the ecotoxicity of 6a, 7a, and 9a to non-target organisms [35]. Briefly, Artemia salina eggs were allowed to hatch in seawater for 48 h at 25 • C. Bioassays were performed in 96well microplates with 15-20 nauplii per well and 200 µL of the compounds test solution. Test solutions were prepared in filtered seawater at concentrations of 25 and 50 µM. All tests included K 2 Cr 2 O 7 as positive control and DMSO as a negative control. Bioassays run in the dark at 25 • C, and the percentage of mortality was determined after 48 h of exposure.

In Silico Evaluation of LogKow
Compounds are considered potentially bioaccumulative if the LogKow (octanol-water partition coefficient) is higher than 3. Therefore, the LogKow value is used as an indicator of the bioaccumulation potential of AF compounds. KOWWIN™ v1.68 (a Log octanol-water partition coefficient calculation program), developed by the United States Environmental Protection Agency (EPA) and the Syracuse Research Corporation (SRC) [39] was used for the in silico calculation of LogKow of the most active compounds in this study.

Statistical Analysis
Data from anti-settlement, antibacterial, and anti-microalgal bioassay were analyzed by one-way analysis of variance (ANOVA), followed by a multi-comparisons Dunnett's test against negative control. For all of the bioassays, the half-maximum response concentration (EC 50 ) values for each compound, when applicable, were calculated using probit regression analysis. Significance was considered at p < 0.01, and 95% lower and upper confidence limits (95% LCL; UCL). The software IBM SPSS Statistics 26 (Armonk, New York, NY, USA) was used for statistical analysis.

Conclusions
Considering the biological potential of phenyl ketones and 1,2,3-triazole ring, in this work 14 acetophenone-1,2,3-triazole hybrids containing different substitution patterns were synthesized and tested for their AF activity in both macro-and microfouling species. Among them, three compounds (6a, 7a, and 9a), containing methoxy groups in the phenyl ketone core with different substituents linked to the heterocyclic ring, revealed to be the most promising compounds against mussel larvae, with EC 50 values lower than 25 µg·mL −1 , while acetophenones 3b, 4b, and 7b showed some inhibitory effect against the growth of biofilm-forming bacteria Roseobacter litoralis. In addition to the activity on macrofouling species, compound 7a also showed AF activity against the microalgae Navicula sp. (EC 50 = 26.73 µM, 8.96 µg·mL −1 ), suggesting a complementary action of this compound against macro-and microfouling species. The most promising compounds of this study (7a and 9a) were also shown to be non-toxic against the non-target species Artemia salina, as well as low bioaccumulative potential. The overall results highlight 7a and 9a as promising compounds, which could be considered hits for the development of effective and eco-friendly AF compounds.