WO2023053059A1 - Antifouling compound, method and uses thereof - Google Patents

Abstract

The present disclosure relates to a synthetic antifouling compound, compositions, methods and uses thereof. The present disclosure further relates to the use of said compound as antifouling agent and compositions thereof, such as coatings and/or paints for surface protection of underwater surfaces.

Description

D E S C R I P T I O N

ANTI FOULI NG COMPOUND, METHOD AND USES TH EREOF

TECHNICAL FIELD

[0001] The present disclosure relates to synthetic antifouling compounds, compositions, methods and uses thereof. The present disclosure further relates to the use of said compounds as antifouling agents and their incorporation in matrices such as coatings and/or paints for underwater surface protection.

BACKGROUND

[0002] The adhesion and growth of microorganisms and/or macroorganisms on surfaces, particularly in contact with aqueous media, is one of the most serious problems in a wide range of industrial sectors (e.g. shipping, water purification units, etc.). This undesired bio-attach, known as biofouling, can promote substrate deterioration, systems clogging, and fluids contamination, resulting in costly maintenance and retrofitting consequences.

[0003] Marine biofouling is a natural process that involves the attachment of a large variety of micro- and macroorganisms (bacteria, algae, invertebrates) in underwater surfaces. When a surface is submerged in water, the biofouling process is sparked by the accumulation and physical adsorption of organic molecules. This conditioning film creates the perfect environment for the settlement and growth of pioneer bacteria, leading to the formation of a biofilm matrix. This initial process leads to the so-called secondary colonization, where a biofilm of multicellular species is formed. Tertiary colonization occurs with the capture of organisms, including larvae of macroorganisms, macroalgae, sponges, cnidarians, polychaetes, molluscs, barnacles, bryozoans, and tunicates.

[0004] Despite this process being quite common in harbours, signalling buoys and ships, it brings serious environmental problems such as the transport of non- indigenous species, leading to biodiversity reduction worldwide and increased fuel consumption and Greenhouse gas emissions due to the increased drag friction caused by the increase of ship's weight and surface roughness.

[0005] Additionally, the most common marine biofouling prevention techniques, which imply the use of toxic and persistent tin-free booster biocides, cause even more environmental problems (Martins, Fillmann et al. 2018). Although the addition of tin- free booster biocides to marine paints has been the most used solution to avoid marine biofouling, they became persistent, bioaccumulative, and toxic (PBT) to the marine ecosystems. Indeed, the use of antifouling (AF) biocides in the aquatic environment has proved to be harmful and several toxic effects were already observed against several non-target marine organisms of several trophic levels (Martins, Fillmann et al. 2018).

[0006] Since recent international regulation (EU Regulation n° 528/2012) banned the use of toxic biocides, efforts have been applied to develop alternative solutions and several natural AF compounds (Liu, Wu et al. 2020) have been discovered. In addition to inhibiting the settlement of biofouling species without causing mortality, new AF agents should also be compatible with commercial marine coatings. Incompatibility with polymeric coatings and commercial supply issues bring many limitations regarding the implementation of natural products in AF marine coatings (Pei and Ye 2015).

[0007] To overcome these limitations, new nature-inspired AF agents obtained by synthesis with high potential to be incorporated in marine coatings were studied (Almeida, Correia-da-Silva et al. 2017). Recently, gallic acid persulfate (GAP, 1), a nontoxic water-soluble nature-inspired AF compound (EC50 = 17.65 pM; LC50/EC50 = 26.61) (Almeida, Correia-da-Silva et al. 2017) was successfully immobilized in marine coatings, leading to non-release coatings (Vilas-Boas, Carvalhal et al. 2020).

[0008] Among the specific metabolic processes used to infer the AF targets of new AF agents, acetylcholinesterase (AChE) and tyrosinase (Tyr) are the most common. Inhibition of AChE interrupts cholinergic signaling, thereby blocking neurotransmission and reducing the success of settlement of fouling organisms (Chen and Qian 2017). This enzyme is the target of Sea Nine 211®, and pesticides, heavy metals and organotin compounds with neurotoxic effects. Another enzyme, Tyr, is implicated in the formation of adhesive plaques in mussels (Chen and Qian 2017). [0009] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

GENERAL DESCRIPTION

[0010] The present disclosure relates to a compound of general formula (I) or an acceptable salt, a hydrate, a solvate, an enantiomer, an atropisomer, a polymorph or an ester thereof

wherein Ri, R2, R3, R4, are independently selected;

Ri is selected from a group consisting of NH2, NHs⁺, amine protecting group, -N- azole;

R2 is selected from a group consisting of H, alkyl;

R3 is selected from a group consisting of H, alkyl, alkyltriazolefluoroaryl;

R4 is selected from a group consisting of H, alkyl; and wherein n is an integer from 1 to 3. .

[0011] For the scope and interpretation of the present disclosure, "amine protecting group" refers to a moiety that temporarily blocks an amine-reactive site in a compound. In an embodiment, an amine protecting group is selectively removable by a chemical reaction.

[0012] In an embodiment, the amine protecting group is selected from a group consisting of carbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, benzylideneamine, p-toluenesulfonamide. In a further embodiment, carbamate protecting groups include, without limitation, fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate, methyl carbamate, ethyl carbamate, 2,2,2-trichloroethyl carbamate, 2-(trimethylsilyl)ethyl carbamate, 1,1- dimethyl-2,2,2-trichloroethyl carbamate, p-methoxybenzyl carbamate, p- nitrobenzylcarbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, and 2,4- dichlorobenzyl carbamate.

[0013] In a preferred embodiment, Ri may be an NHs⁺ or t-butyl carbamate.

[0014] In a preferred embodiment, F or FU may be C1-C6 alkyl or H.

[0015] In a preferred embodiment, FU or F may be methyl or H.

[0016] In a preferred embodiment, FU and F may be equal.

[0017] In a preferred embodiment, the salt may be a fluoride, chloride, bromide, iodide, acetate, citrate, maleate, or mesylate.

[0018] In a preferred embodiment, the compound may be one of the follows:

(Compound 26);

(Compound 31).

[0019] Another aspect of the present disclosure relates to a composition comprising the compound disclosed in the present disclosure.

[0020] In a preferred embodiment, the composition may comprise 0.1 to 10 wt.% of the compound of the present disclosure, preferably from 1 to 4 wt.% of the compound of the present disclosure; more preferably from 1 to 2 wt.% of the compound of the present disclosure.

[0021] In a preferred embodiment, the composition may be incorporated in a polymeric formulation used to obtain articles such as films, yarn, stent, devices or part of devices; preferably wherein said article may be a fish net or a medical device.

[0022] In a preferred embodiment, the composition may be a coating composition.

[0023] In a preferred embodiment, the composition may be an antifouling paint or varnish composition for protecting underwater surfaces, in particular surfaces submerged in a marine environment.

[0024] In a preferred embodiment, the composition may further comprise one of the following additives: dye, polymer, filler, essential oil, stabilizer, surfactant, crosslinker agent, curing agent, biocides, solvent, or mixtures thereof.

[0025] In a preferred embodiment, the composition may be a coating, preferably a solvent-based paint.

[0026] In a preferred embodiment, the composition may be a polymeric paint composition, preferably a polyurethane based paint.

[0027] Another aspect of the present disclosure relates to the use of the disclosed compound/composition as an antifouling agent, preferably as a marine antifouling agent (macro or micro antifouling agent); in particular the use of compounds

[0028] In a preferred embodiment, the compound or composition may further be used as an micro antifouling agent, in particular the use of compound

[0029] Another aspect of the present disclosure relates to an article comprising the composition or compound of the present disclosure; preferably wherein said article is a paint, a varnish, a stone, a boat, a surfboard, a net (preferably fish net, aquaculture net), a buoy, or a medical device.

[0030] Another aspect of the present disclosure relates to a process for obtaining the disclosed compounds by reacting, by scheme 1 or sheme 2, a compound of general formula (II)

wherein Ri, R2, R3, are independently selected;

Ri and R3 are an alkyl; preferably C1-C6 alkyl;

R2 is selected from a group consisting of H, alkyl; preferably C1-C6 alkyl. [0031] In an embodiment, the process comprises a step of reacting an amine, according to scheme 2, with the compound of general formula (II), wherein Ri, R2, R3, are independently selected; Ri, R2, R3 are selected from a group consisting of a H, alkyl; preferably C1-C6 alkyl.

[0032] In an embodiment, the process comprises the following steps: reacting the compound of general formula (II) with a bromine containing a terminal alkyne, wherein Ri, or R2, or R3, is H; reacting with an amine; and reacting the terminal akyne with an azide, as represented in scheme 1.

[0033] In another embodiment, the process further comprises a step of removing the alkyl groups (Ri, R2 or R3).

BRI EF DESCRIPTION OF TH E DRAWINGS

[0034] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of the invention.

[0035] Figure 1: Design of potential new antifouling compounds.

[0036] Figure 2: Embodiment of the anti-settlement activity of compounds 2-31 (at 50 pM) towards larvae of the mussel Mytilus galloprovincialis after 15 h of exposure. B - Negative control (Natural sterilized seawater with DMSO); C - Copper sulfate (CuSC ) at 5 pM.

[0037] Figure 3A: Embodiment of A - ¹H NMR spectra of compound 9 (CDCI3, 300.13 MHz) .

[0038] Figure 3B: ¹³C NMR spectra of compound 9 (CDCI3, 1 A1 MHz).

[0039] Figure 3C: HRMS spectra of compound 9

[0040] Figure 4A: Embodiment of A - ¹H NMR spectra of compound 25 (DMSO-de, 400.15 MHz); .

[0041] Figure 4B: B - ¹³C NMR spectra of compound 25 (DMSO-d₆, 100.62 MHz)

[0042] Figure 4C: C - HRMS spectra of compound 25 [0043] Figure 5A: Embodiment of A - ¹H NMR spectra of compound 26 (DMSO-de,

300.13 MHz).

[0044] Figure 5B: B - ¹³C NMR spectra of compound 26 (DMSO-d₆, 75.47 MHz)

[0045] Figure 5C: C- HRMS spectra of compound 26

[0046] Figure 6A: Embodiment of A - ¹H NMR spectra of compound 31 (DMSO-de, 400.15 MHz).

[0047] Figure 6B: B - ¹³C NMR spectra of compound 31 (DMSO-d₆, 75.47 MHz)

[0048] Figure 6C: C - HRMS spectra of compound 31

[0049] Figure 7: Embodiment of in vitro acetylcholinesterase (AChE) activity in the presence of compounds 9, 11, 18, 25, 26, and 31. B: ultra-pure water control; C+: positive control with eserine at 100 and 200 pM.

[0050] Figure 8: Embodiment of results of (A) biofilm prevention and (B) biofilm reduction assays with different concentrations of compound 26. Letters were assigned in alphabetic order from the highest to the lowest value (from a to d). These assignments were made as long as statistically significant differences existed between the number of biofilm cells with a confidence level greater than 95% (p < 0.05). The means ± SDs for three independent experiments are illustrated.

[0051] Figure 9: Anti-settlement activity of compound 26 (2.04 ± 0.08 wt. %) directly incorporated in a polyurethane (PU)-based coating (26-PU) towards plantigrades of the mussel M. galloprovincialis. Negative control: PU-based coating free of bioactive agent (control-PU).

[0052] Figure 10: Embodiment of the effect of PU-based coatings containing different concentrations of compound 26 and crosslinker (CL) on biofilm development of Pseudoalteromonas tunicata for 49 days. The analysed parameter refers to the number of biofilm cells. Letters were assigned in alphabetic order from the highest to the lowest value (from a to c) for each time point. These assignments were made as long as statistically significant differences existed between the biofilms with a confidence level greater than 95% (p < 0.05). The means ± SDs for two independent experiments are illustrated. [0053] Figure 11: Representative biofilm structures of Pseudoalteromonas tunicata on surface treated with compound 26 (1 wt.%)-PU based coating (1% Compound 26/PU), surface treated with compound 26 (2 wt.%)-PU based coating (2% Compound 26/PU), and surface treated with compound 26 (2 wt.%)-triaziridine propionate crosslinker (TZA)/PU based coating (2% Compound 26/PU/CL), after 49 days of biofilm formation. Images were obtained from confocal z-stacks using IMARIS software and present an aerial view of the biofilms (shadow projection on the right). The white scale bar corresponds to 50 pm.

[0054] Figure 12: Embodiment of the biofilm structural parameters obtained from the z-stacks acquired at the confocal laser scanning microscopy (CLSM) after 49 days: biovolume (A) and thickness (B). Letters were assigned in alphabetic order from the highest to the lowest value (from a to c). These assignments were made as long as statistically significant differences existed between the biofilms with a confidence level greater than 95% (p < 0.05). The means ± SDs for two independent experiments are illustrated.

DETAILED DESCRIPTION

[0055] The present disclosure relates to a synthetic antifouling compound, preferably marine antifouling, compositions, methods and uses thereof. The present disclosure further relates to the use of the said compound as an antifouling agent and their incorporation in matrices such as coatings and/or paints for underwater surface protection.

[0056] Gallic acid persulfate (GAP, compound 1), with formula (III), was identified as a lead compound and further modified to obtain a compound with suitable AF properties.

A series of new 16 derivatives of compound 1, was synthesized following the molecular modification approaches presented in Figure 1, to improve potency and lipophilicity while maintaining low toxicity. An example, the substitution of sulfate with other groups that could retain the antifouling activity was envisioned to decrease water solubility of compound 1 and facilitate the scale-up process. Previous research showed that the presence of hydroxyls instead of the sulfate groups in compound 1 produced an inactive compound with formula (IV)(gallic acid) (Almeida, Correia-da-Silva et al. 2017).

[0057] The presence of a triazole ring was envisioned to improve potency and lipophilicity. Triazoles are present in several drugs, for example in the so-called "azole" family of antifungals, and are commonly used in Medicinal Chemistry to increase oral bioavailability due to their chemical stability towards oxidation and acid hydrolysis (Tron, Pirali et al. 2008).

[0058] In an embodiment, the octanol-water partition coefficient (Log K_ow) was calculated. All the planned compounds have a low tendency to bioaccumulate in organisms' tissues (Log K_ow< 3).

[0059] In an embodiment to assess the AF effectiveness and toxicity of the synthesized derivatives of compound 1, in vivo bioassays using adhesive larvae of the most common macrofouling species, Mytilus galloprovincialis, were conducted and the EC50 and LC50 were further calculated for the most potent compounds. After the selection of the most promising AF compounds, putative AF target mechanisms were studied through the evaluation of acetylcholinesterase (AChE) and tyrosinase (Tyr) activities. Studies concerning the antimicrofouling activity, namely the antibiofilm activity using Pseudoalteromonas tunicata, were also conducted for one of the disclosed AF compounds. In a further embodiment, ecotoxicity for two non-target marine organisms, nauplii of the crustacean Artemia salina and the marine microalgae Phaeodactylum tricornutum, was evaluated. At last, a polyurethane (PU)-based marine coating comprising one of the disclosed AF compounds was conceived to evaluate its ability to reduce the settlement of mussel larvae and biofilm formation by a marine bacteria after incorporation in a coating formulation.

[0060] To synthesize a library of GAP (1) derivatives, the synthesis of triazole derivatives of syringic (2) and sinapic acid (11) was designed (Scheme 1). The first step to obtaining triazole-linked derivatives was the propargylation of compound 2 and compound 11 with propargyl bromide, in the presence of anhydrous CsCCh, which gave compound 3 and 12 in 78 % and 72 % yield, respectively. As the propargyl bromide also reacted with the carboxylic acid of both compounds 2 and 11, removal of propargyl bromide at this position was necessary. Hydrolysis of compound 3 and 12 was achieved at 70 °C, through basic hydrolysis (NaOH 1 M) for compound 3 and in the presence of acid (HCI) for compound 11 due to its susceptibility to undergo addition in the presence of a base due to the double bond (Scheme 1).

[0061] Scheme 1 depicts the synthesis of derivatives 3-10 (A); the- synthesis of derivatives 12-19 (B). THF - tetrahydrofuran; TBTU - 2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium tetrafluoroborate; TEA - triethylamine; TFA - trifluoracetic acid; rt - room temperature.

[0062] For the scope and interpretation of the present disclosure, it is defined that "room temperature" should be regarded as a temperature between 15-30 °C, preferably between 18-25 °C, more preferably between 20-22 °C.

[0063] Scheme 1:

[0064] Scheme 1 (continued):

[0065] A great variety of coupling reagents are available in the state of the art to improve the reaction between a carboxylic acid and an amine. In an embodiment, 2- (l/7-benzotriazole-l-yl)-l,l,3,3-tetramethylaminium tetrafluoroborate (TBTU) was used to obtain the disclosed compounds. Compounds 4 and 13 were allowed to react with triethylamine (TEA) and TBTU, for 15 min, after which /V-(tert-butoxycarbonyl)- 1,2-diaminoethane was added (Scheme 1). Compounds 5 and 14 were obtained in 45 % and 55 % yield, respectively.

[0066] Following, the copper(l)-catalyzed 1,3-dipolar cycloaddition of alkynes 5 and 14 with several azides was accomplished in the presence of sodium ascorbate and CUSO4-5H2O, in a mixture of THF and water, at room temperature (Scheme 1), to obtain compounds 6-9 and 15-18, with 16 - 83 % yield.

[0067] In an embodiment, deprotected amines 10 and 19 were obtained, after allowing to react amines 9 and 18 with trifluoracetic acid (TFA) at room temperature, in 49 % and 50 % yield, respectively.

[0068] In another embodiment, different substituents containing amine groups were also introduced in trimethoxybenzoic acid (20) through an amide bond (Scheme 2). Compound 20 is commercially available at a low cost and was selected as the starting material for this reaction. By using this compound, it was possible to increase selectivity, since attempts using gallic acid originated several products due to the presence of three phenolic groups. Briefly, compound 20 reacted with TEA and TBTU, in THF, for 15 min, after which the respective amine (21-24) was added (Scheme 2). The reaction proceeded at room temperature for 0.5 to 24 h, and compounds 25-28 were obtained in moderate to good yields (18-84 %).

[0069] Scheme 2 depicts the synthesis of amide derivatives 21-25 through a coupling reaction and derivatives 26-31 using BBrj. TBTU - 2-(lH-Benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium tetrafluoroborate; TEA - Triethylamine; THF - Tetrahydrofuran; rt - room temperature.

[0070] Scheme 2:

[0071] In an embodiment, compounds 21-25 reacted with BB at -40 °C for 30 min and overnight at room temperature to give derivatives 26-30 in 21-83 % yield (Scheme 2). For amides 21-23, not only the methoxyl groups were removed but also the protecting group Boc, which is unstable in acid conditions, forming the bromide salt of the amine (26-28). When amide 21 reacted with only 0.7 equivalents/OCHs of BB compound 31 was obtained, in which only the Boc group was removed. Overall, the reactions proceeded well with complete conversion of the starting material in one major product.

[0072] In an embodiment, for compounds 26-29, after completion of the reaction, the solid formed after the addition of BBrs was filtrated and dissolved in water to cleave the aryloxydibromoborane derivative following dialysis to remove by-products of the reaction. After lyophilization, the presence of B(OH)3 was noted for compounds 26-31, as detected by Fourier-transform infrared spectroscopy (FTIR) measurements with two intense bands appearing around 1450 and 1195 cm⁴. In an embodiment, isopropanol was used to precipitate B(OH)s and the desirable compounds were obtained after evaporation of the filtrate. Compounds 30 and 31 were isolated by filtration after precipitation with water and the IR spectra did not show any bands corresponding to the presence of B(OH)s.

[0073] In an embodiment, the synthesis processes can be described as follows:

[0074] Solvents were of analytical grade and were purchased from Sigma-Aldrich (Spain). Syringic acid (2, 60014), sinapic acid (11, D2932), /V-(tert-butoxycarbonyl)-l,4- diaminobutane (1373), and /V-(tert-butoxycarbonyl)-l,2-diaminoethane (A1371) were purchased from TCI (Zwijndrecht, Belgium); CsCCh (441902), propargyl bromide solution 80 wt % in toluene (P51001), sodium ascorbate (A7631), ~0.5 M solution of 1- azido-4-bromobenzene in tert-butyl methyl ether (779377), ~0.5 M solution of 1-azido- 4-chlorobenzene in tert-butyl methyl ether (727482), ~0.5 M solution of 4- (azidomethyl) benzonitrile in tert-butyl methyl ether (745316), ~0.5 M solution of 1- azido-4-fluorobenzene in tert-butyl methyl ether (779253) and TFA (T6508f), trimethoxybenzoic acid (20, T69000), boron tribromide (BB , 202207), 2-(l/7- benzoimidazol-2-yl) ethylamine (CDS013165) and /V-phenylethylenediamine (P2415) were purchased from Sigma-Aldrich (Spain); TBTU (303180250) and N- methylpyrrolidone (99.5%) were purchased from AcrosOrganics (Geel, Belgium), TEA (489556) was purchased from Carlo Erba (France) and CuSC -SHzO (ChemLab CLOO.1127.1000) was purchased from ChemLab (Zedelgem, Belgium). Spectra/Por Dialysis membranes (MWCO 100-500 Da) were purchased from Spectrum Laboratories, Inc. (California, USA). Sodium hydrogen carbonate (NaHCCh) and hydrochloric acid (HCI) were purchased from VWR Chemicals (Portugal).

[0075] TLC separations were performed using Merck silica gel 60 (GF254) plates, and flash column chromatography separations were performed using Fluka silica gel 60 (0.04-0.063 mm). Melting points were obtained using a Kbfler microscope and are uncorrected. Infrared spectra were recorded in a KBr microplate in a FTIR spectrometer Nicolet iSlO from Thermo Scientific (U.S.A.) with Smart OMNI- Transmission accessory (Software OMNIC 8.3). ¹H and ¹³C Nuclear magnetic resonance (NMR) spectra were acquired in CDCI3 or DMSO-de at room temperature either on a Bruker Avance 300 (300.13 MHz for ^TH and 75.47 MHz for ¹³C NMR) or 400 (400.14 MHz for ¹H and 100.64 MHz for ¹³C NMR) instrument. Chemical shifts are expressed in 6 (ppm) values relative to tetramethylsilane (TMS) as an internal reference. High- resolution mass spectrometry (HRMS) was performed on an LT OrbitrapTM XL hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0. at CEMUP— University of Porto, Portugal, on a Bruker FTMS APEX III mass spectrometer (Bruker Corporation, Billerica, MA, USA) and recorded as electrospray ionization (ESI) mode in Centro de Apoio Cientifico e Tecnoloxico a Investigacion (CACTI, University of Vigo, Pontevedra, Spain) and on a Q Exactive Focus Hybrid Quadrupole Orbitrap Mass Spectrometer (Thermo Fisher Scientific), controlled by Q Exactive Focus (Exactive Series) 2.9 and Thermo Scientific Xcalibur 4.1.31.9 software.

- General procedure for the synthesis of derivatives 3 and 12

[0076] To a solution of 2 (syringic acid, TCI 60014, lg, 5.1 mmol) or 11 (sinapic acid, TCI D2932, lg, 4.5 mmol) and CsCO₃ (Aldrich 441902, 2.2-2.5 g, 6.7-7.6 mmol) in 20 mL of dry acetone (Biosolve), a propargyl bromide solution (Aldrich P51001, 80 wt. % in toluene, 1.0-1.6 mL, 6.7-7.6 mmol) was added. The mixture was kept under reflux for 3 to 6 h and after cooling the CsCO₃ was filtered. After concentration under reduced pressure, the residue was dissolved in CH2CI2 and washed twice with H2O. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure. The residue was further purified by crystallization with methanol (MeOH) to afford white crystals.

[0077] Synthesis of 3,5-dimethoxy-4-(prop-2-yn-l-yloxy)benzoic acid (4): To a solution of prop-2-yn-l-yl 3,5-dimethoxy-4-(prop-2-yn-l-yloxy)benzoate (3, 0.2 g, 0.73 mmol) in a mixture of tetrahydrofuran (THF)/MeOH (10 mL, 1:1), an aqueous solution of NaOH IM (3mL) was added. The mixture was heated to 70 °C for 1 h. After cooling to room temperature, the mixture was cooled to 0 °C in an ice bath. The mixture was diluted with H2O (lOmL) and a solution of HCI IM was added and a solid precipitated. The white solid was filtered and dried under vacuum (0.135 g, 0.57 mmol, 78 % yield). IR (KBr) u_max: 3442, 3271, 2966, 2658, 2126, 1692, 1593, 1468, 1420, 1340, 1287, 1226, 1181, 1137, 998, 860, 766, 726, 674 cm ¹; ^TH NMR (DMSO-d₆, 300.13 MHz) <5:13.00 (1H, brs, COOH), 7.23 (2H, s, H-2',6'), 4.69 (2H, d, J= 2.4 Hz, H-l”), 3.82 (6H, s, 3',5'-OCH₃), 3.46 (1H, t, J= 2.4 Hz, H-3") ppm; ¹³C NMR (DMSO-d₆, 75.47 MHz) <5: 166.9 (C-l), 153.0 (C-3',5'), 138.6 (C-4'), 127.1 (C-l'), 106.4 (C-2', 6'), 79.4 (C-2"), 78.0 (C-3"), 59.2 (C-l”), 56.0 (3',5'-OCH₃) ppm; HRMS (ESI/TOF) m/z: [M+Na]⁺ calcd for Ci₂Hi₂O₅Na 259.05769, found 259.05766.

Synthesis of (E)-3-(3,5-dimethoxy-4-(prop-2-yn-l-yloxy)phenyl)acrylic acid (13)

[0078] To a solution of prop-2-yn-l-yl (£)-3-(3,5-dimethoxy-4-(prop-2-yn-l- yloxy)phenyl)acrylate (12, 0.72 g, 2.4 mmol) in THF:H₂O (2:1, 15 mL) HCI cone (5 mL) was added. The mixture was heated to 70 °C for 5 h. After cooling, THF was evaporated, and the H₂O was extracted twice with CH₂CI₂. The organic phase was dried over Na₂SO4 anhydrous and filtered. The residue obtained was further purified with an anion exchange resin (Amberlite IRA-93) in MeOH and a white solid was obtained (0.42 g, 1.6 mmol, 67 % yield). IR (KBr) u_max: 3432, 3275, 3015, 2944, 2841, 2652, 2360, 2127, 1686, 1625, 1586, 1504, 1454 1403, 1343, 1287, 1245, 1204, 1161, 1122, 998, 983, 832, 674, 623 cm ¹; ^TH NMR (DMSO-d₆, 300.13 MHz) <5: 7.52 (1H, d, J= 15.9 Hz, H- 3). 7.04 (2H, s, H-2',6'), 6.56 (1H, d, J= 15.9 Hz, H-2). 4.63 (2H, d, J= 2.5 Hz, H-l”), 3.81 (6H, s, H-3', 5'), 3.44 (1H, t, J= 2.4 Hz, H-3”) ppm; ¹³C NMR (DMSO-d₆, 75.47 MHz) <5: 167.8 (C-l), 153.3 (C-3',5'), 144.0 (C-3), 136.5 (C-4'), 130.5 (C-l'), 118.9 (C-2), 105.7 (C- 2', 6'), 79.6 (C-2”), 77.9 (C-3”), 59.2 (C-l”), 56.1 (3',5'-OCH₃) ppm; HRMS (ESI-) m/z: [M-H]- calcd for CI₄HI₃O₅ 261.0768, found 261.0771.

General procedure for the synthesis of derivatives 5 and 14

[0079] To a solution of 3,5-dimethoxy-4-(prop-2-yn-l-yloxy)benzoic acid (4, 1.0 g, 4.2 mmol) or (£)-3-(3,5-dimethoxy-4-(prop-2-yn-l-yloxy)phenyl)acrylic acid (13, 0.3 g, 1.14 mmol) in 10-20 mL of THF, TBTU (AcrosOrganics 303180250, 0.55-2.04 g, 1.7-6.4 mmol) and TEA (Carlo Erba 489556, 0.08-0.3 mL, 0.57-2.1 mmol) were added. After 15 min under stirring, /V-(tert-butoxycarbonyl)-l,2-diaminoethane (TCI A1371, 0.2-0.8 mL, 1.4-5.1 mmol) was added. The reaction was completed after 2 h. THF was evaporated until dryness and the residue was dissolved in ethyl acetate, extracted twice with an aqueous solution HCI 1 M, twice with a saturated solution of NaHCO₃, and washed twice with water. The organic phase was dried over NazSC and the solid was further purified by crystallization with acetone.

General procedure for the synthesis of triazole derivatives 6-9 and 15-18

[0080] To a solution of tert-butyl (2-(3,5-dimethoxy-4-(prop-2-yn-l- yloxy)benzamido)ethyl)carbamate (5, 0.10-0.15 g, 0.260-0.396 mmol) or tert-butyl (£)- (2-(3-(3,5-dimethoxy-4-(prop-2-yn-l-yloxy)phenyl)acrylamido)ethyl)carbamate (14, 0.051-0.10 g, 0.126-0.250 mmol) in 10 mL of THF, a solution of sodium ascorbate (Sigma A 7631, 0.041-0.074 g, 0.208-0.320 mmol) and CuSO₄-5H₂O (ChemLab CLOO.1127.1000, 0.024-0.060 g, 0.151-0.320 mmol) in 5 mL of water was added.

[0081] tert-Butyl (2-(4-((l-(4-fluorophenyl)-lH-l,2,3-triazol-4-yl)methoxy)-3,5- dimethoxybenzamido)ethyl)carbamate (9): After 10 min under stirring, a ~0.5 M solution of l-azido-4-fluorobenzene in tert-butyl methyl ether (0.74 mL, 0.32 mmol) was added, and the reaction mixture was left under room temperature for 24 h. THF was evaporated and the residue was diluted with water and extracted twice with CH2CI2. The organic phase was dried over anhydrous NazSO₄, filtered and evaporated until dryness. The solid obtained was further purified through flash column chromatography (100 % CHCI3 to 8:2 CHCl3:acetone) to give a white solid (0.113 g, 0.22 mmol, 83 % yield); IR (KBr) u_max: 2248, 3288, 3145, 3083, 2979, 2936, 2872, 2838, 1690, 1637, 1585, 1545, 1519, 1499, 1545, 1519, 1499, 1463, 1347, 1286, 1234, 1178, 1130, 970, 848, 668, 609 cm ¹; ^TH NMR (CDCI3, 300.13 MHz) <5: 8.10 (1H, s, H-triazole), 7.72-7.67 (2H, m, H-PhF), 7.49 (1H, brd, NH amide), 7.25-7.19 (2H, m, H-PhF), 7.11 (2H, s, H-2',6'), 5.30 (2H, s, CH₂-triazole), 5.04 (1H, brd, NHBoc), 3.90 (6H, s, 3',5'-OCH₃), 3.57-3.52 (2H, m, CH2CH2), 3.40 (2H, m, CH2CH2), 1.40 (9H, s, Boc) ppm; ¹³C NMR (CDCI3, 75.47 MHz) <5: 171.6 (C=O amide), 165.7 (C-F, d, J= 223.0 Hz), 160.9 (C=O Boc), 153.1 (C-3',5'), 145.9 (C-triazole), 139.0 (C-4'), 133.5 (C-PhF), 130.3 (C-l'), 122.7 (C-PhF, d, J= 8.7 Hz), 121.5 (C-H triazole), 116.9 (C-PhF, d, J= 23.1 Hz), 104.4 (C-2',6'), 66.5 (OCHz-triazole), 56.4 (3',5'-OCH₃), 42.9 (CH₂), 40.3 (CH₂), 28.5 (C(CH₃)₃) ppm; HRMS (ESI+) m/z: [M+H]⁺ calcd for C25H₃IFN₅O₆516.2253, found 516.2261. General procedure for the synthesis of derivatives 10 and 19

[0082] To a solution of derivative 9 (0.08 g, 0.160 mmol) or 18 (0.09 g, 0.166 mmol) in 5 or 7 mL of CH2CI2 or THF, respectively, TFA (Sigma T6508f, 2 mL, 26.1 mmol) was added. The mixture was kept under stirring at room temperature for 1-3 h. The reaction was extracted with a saturated aqueous solution of sodium bicarbonate. A solid precipitated between the two phases and was filtered through vacuum to afford a white solid.

General procedure for the synthesis of derivatives 21 -25

[0083] To a solution of trimethoxybenzoic acid (20, Aldrich T69000, 0.1 - 2 g, 0.47-9.4 mmol) in THF (10-40 mL), TBTU (TCI B1658, 0.23-6.1 g, 0.71-18.9 mmol) and TEA (Carlo Erba 489556, 0.03-0.7 mL, 0.24-4.7 mmol) were added.

[0084] A/-(2-(lH-benzo[d]imidazol-2-yl)ethyl)-3,4,5-trihydroxybenzamide (25): After 15 min under stirring, 2-(l/7-benzoimidazol-2-yl) ethylamine (0.25 g, 1.55 mmol) was added. The reaction was left overnight, with stirring. The solvent was evaporated until dryness and the residue was dissolved in ethyl acetate, extracted twice with a sat sol NaHCCh, and washed twice with water. The organic phase was dried over sodium sulfate. The residue obtained after evaporation of the organic solvent was further purified by crystallization with ethyl acetate to afford white crystals (0.075 g, 0.21 mmol, 18 % yield). IR (KBr) u_ma_X: 3447, 3284, 3096, 3051, 2919, 2849, 1636, 1583, 1553, 1502, 1457, 1429, 1337, 1243, 1124, 749 cm ¹; ^XH NMR (DMSO-d₆, 400.15 MHz) <5: 12.30 (1H, brs, NH benzimidazole), 8.63 (1H, t, J= 5.6 Hz, NH amide), 7.48-7.46 (2H, m, H-benzimidazole), 7.16 (2H, s, H-2',6'), 7.14-7.10 (2H, m, H-benzimidazole), 3.81 (6H, s, 3',5'-OCH₃), 3.74-3.71 (2H, m, CH2CH2), 3.69 (3H,s, 4'- OCH₃), 3.11-3.07 (2H, m, CH2CH2) ppm; ¹³C NMR (DMSO-d₆, 100.62 MHz) 6: 165.8, 152.9, 152.5, 139.9, 129.7, 121.2, 104.8, 79.3, 79.0, 78.6, 60.1, 55.9, 38.1, 28.9 ppm; HRMS (ESI+) m/z: [M+H]⁺ calcd for C19H22N3O4 356.1605, found 356.1605.

General synthesis of deprotected amide derivatives 26-31

[0085] To a solution of protected amine (derivatives 21-25, 0.08-lg, 0.23-2.8 mmol) in anhydrous CH2CI2 (10-55 mL) at -40 °C, a BBrs solution (~1M in CH2CI2, 1.6-2.1 mL, 12.8 mmol) was added. The mixture was kept at -40°C for approximately 30 min. The solid formed was filtrated, washed with CH2CI2 and dried.

[0086] 2-(3,4,5-Trihydroxybenzamido)ethan-l-aminium bromide (26): The obtained solid was dissolved in water and a dialysis was performed. Water was removed by lyophilization and the solid obtained was dissolved in a small amount of hot isopropanol. The solid was filtrated, and the filtrate was evaporated to afford the desired product as a pale white solid (0.68 g, 2.3 mmol, 83 % yield). IR (KBr) Umax: 3381, 3342, 3211, 2986, 2958, 1608, 1522, 1505, 1343, 1320, 1276, 1029, 932, 757 cm ¹; ^XH NMR (300 MHz, DMSO-d₆) 6: 9.07 (2H, brs, 3', 5' -OH), 8.76 (1H, brs, 4' -OH), 8.20 (1H, t, J= 5.6 Hz, NH amide), 7.75 (3H, brs, NH₃ ⁺), 6.85 (2H, s, H-2',6'), 3.43 (2H, q, J= 5.8 Hz, CH₂), 2.94 (2H, q, J= 5.8 Hz, CH₂) ppm; ¹³C NMR (DMSO-d₆, 75.47 MHz) 6: 167.1 (C=O), 145.5 (C-3',5'), 136.5 (C-4'), 124.2 (C-l'), 106.9 (C-2',6'), 37.1 (CH₂CH₂) ppm; HRMS (ESI- ) m/z: [M-H]’ calcd for C9Hi2BrN₂O₄ 290.9986, found 290.9989.

[0087] 2-(3,4,5-Trimethoxybenzamido)ethan-l-aminium bromide (31): The yellow solid formed after adding BBr₃ was filtrated, washed with CH2CI2 and dried (0.75 g, 2.24 mmol, 79 % yield). IR (KBr) u_max: 3416, 3219, 2974, 2839, 2506, 2261, 1634, 1587, 1549, 1501, 1449, 1335, 1237, 1184, 1128, 985, 857, 779, 763, 748, 648 cm ¹; ^TH NMR (DMSO-d₆, 400.14 MHz) <5: 8.66 (1H, t, J= 5.6 Hz, NH-amide), 7.87 (3H, brs, NH₃ ⁺), 7.23 (2H, s, H-2',6'), 6.48 (1H, brs, NH-amide), 3.83 (6H, s, 3',5'-OCH₃), 3.70 (3H, s, 4'-OCH₃), 3.51 (2H, q, J= 6.2 Hz, CH₂), 3.00 (2H, q, J= 5.9 Hz, CH₂) ppm; ¹³C NMR (CDCI3, 100.62 MHz) <5: 166.5 (C=O amide), 152.6 (C-3',5'), 140.2 (C-4'), 129.2 (C-l'), 105.1 (C-2',6'), 60.2 (4'-OCH₃), 56.2 (3',5'-OCH₃), 37.2 (CH₂CH₂) ppm; HRMS (ESI-) m/z: [M-H]’ calcd for Ci₂Hi₈BrN₂O₄ 333.0455, found 333.0447.

[0088] In an embodiment, the Mediterranean mussel, M. galloprovincialis, was used as a target species to study the settlement inhibition of the disclosed synthetic compounds using a previously validated anti-macrofouling bioassay. Intermediates 2-5, 11-15, and 20-25 were also included in the screening assay (Figure 2). Briefly, for the screening bioassay, competent M. galloprovincialis plantigrades with exploring behavior (i.e., moving their foot searching for the appropriate substrate to settle) were selected and exposed to compounds 2-31 at a concentration of 50 pM in 24-well microplates and left in an incubator for 15 h, at 18 ± 1 °C, in the darkness (Almeida, Correia-da-Silva et al. 2017). Test solutions were prepared in filtered natural seawater (previously treated by UV light, and carbon filters and mechanically filtered with 0.45 pM filter before use) and obtained by dilution of the compounds stock solutions (50 mM) in DMSO (compounds insoluble in DMSO were solubilized in ultrapure water). Four well replicates were used per condition with five plantigrades per well and 2.5 mL of test solution. A negative control, with natural seawater + DMSO (0.01 %) was included in all bioassays, as well as a positive control with 5 pM CuSO4 (a potent AF agent). At the end of the exposure period, the anti-settlement activity was determined by the presence/absence of byssal threads produced by each individual efficiently attached. Compounds that showed anti-settlement effectiveness at a concentration of 50 pM were selected for further testing at higher and lower successive concentrations (200, 100, 50, 25, 12.5, 6.25, and 3.12 pM) for the determination of the semimaximum response concentrations that inhibited 50% larval settlement (EC50) and the median lethal dose (LC50). Given its potency, compound 26 was also tested at lower successive concentrations of 1.56 and 0.78 pM to obtain its EC50 value.

[0089] At 50 pM, the larvae settlement decreased to less than 35 % in the presence of four compounds 9, 25, 26, and 31. Based on the primary screening, the ECso and LC50 for these compounds were determined (Table 1). Among compounds 26-28, all with primary amines (in bromide salt), a shorter alkyl chain (n=2) was associated with an improvement in activity (derivative 26). When comparing two compounds with the same amine (NHsBr) and size of the alkyl chain (n=2), the hydroxylated compound 26 was more effective than the parent methoxylated derivative, compound 31. Other hydroxylated derivative, compound 29, with an NH-phenyl instead of NHsBr (compound 26), was inactive, highlighting the importance of NHsBr in compound 26. In contrast, when a N-azole was linked to the alkyl chain (compound 25), only the methoxylated derivative was active (compound 25). Table 1 - AF effectiveness versus toxicity of derivatives 9, 25, 26 and 31, towards the anti-settlement of mussel plantigrades.

ECso, minimum concentration that inhibited 50% of larval settlement; LCso, the median lethal dose; LC50/EC50, therapeutic ratio. Note: reference values for ECso< 25 pg/mL (U.S. Navy program) and therapeutic ratio (LC50/EC50) higher than 50.

[0090] In an embodiment, from the four selected compounds (Table 1), three (9, 26, and 31) showed ECso lower than the lead compound, gallic acid persulfate (formula II). ECso of gallic acid persulfate was previously described (Almeida, Correia-da-Silva et al. 2017) using the same experimental conditions. Compound 9 has a triazole moiety that links the dimethoxybenzene moiety to a fluorobenzene ring, while compounds 26 and 31 are derivatives of gallic acid with the presence of three phenols or methoxyl groups at the benzene moiety, respectively. Compound 26 was the most potent compound being able to inhibit the settlement of mussel larvae in a concentration seven-fold lower than the described in the literature for lead compound (Almeida, Correia-da- Silva et al. 2017). More relevant, this increase in potency, was not associated with an increase in toxicity. In fact, compound 26 did not cause mortality to this target species even at the highest concentration tested (200 pM), similarly to what is described for the lead compound 1 (Almeida, Correia-da-Silva et al. 2017). Therefore, LCso value was considered as higher than 200 pM, and 200 pM was used to estimate the therapeutic ratio (LC50/EC50). Compound 26 also demonstrated a therapeutic ratio much higher than 15, as recommended by U.S. Navy (Kwong, Miao et al. 2006)for promising AF compounds.

[0091] Regarding the calculation of the Log K_ow, compounds 9, 25, 26, and 31 showed lower Log K_ow compared to compound 1, showing a lower potential to bioaccumulate in marine organisms and better potential to be compatible with marine coatings. [0092] In an embodiment, the ability of compounds 9, 25, 26, and 31 to inhibit AChE and Tyr activities was explored. AChE activity was evaluated using Electrophorus electric acetylcholinesterase Type V-S (SIGMA C2888, E.C. 3.1.1.7), according to Ellman, Courtney et al. (1961) with some modifications. Reaction solution containing phosphate buffer IM pH 7.2, dithiobisnitrobenzoate (DTNB) 10 mM (acid dithiobisnitrobenzoate and sodium hydrogen carbonate in phosphate buffer) and acetylcholine iodide 0.075M was added to pure AChE enzyme (0.25 U/mL) and compound 26 (final concentration of 25, 50, and 100 pM) in quadruplicate. All tests included a positive control with eserine and a negative control with DMSO. The optical density was measured at 412 nm in a microplate reader (Biotek Synergy HT, Vermont, USA) for 5 min, at 25 °C. Tyr activity was assessed using Agaricus bisporus tyrosinase (EC 1.14.18.1) according to (Adhikari, Devkota et al. 2008) with appropriate adaptations. The enzymatic reaction follows the catalytic conversion of L-Dopa to dopaquinone and the formation of dopachrome by measuring the absorbance at 475 nm. Briefly, 50 pL of tyrosinase (25 U.mL^-1) in 50 mM phosphate buffer pH 6.5, and compound final concentration of 25, 50, and 100 pM in quadruplicate was added to L - Dopa (25 mM) to trigger the reaction. Kojic acid (100 and 200 pM) was used as positive control and DMSO as a negative control.

[0093] Compound 26 slightly inhibited the AChE activity (35%) at a concentration nearly forty-fold higher (100 pM, Figure 7) than the anti-settlement EC50. No inhibition of Tyr activity was observed for the compounds tested. Overall, these two pathways were not significantly affected after exposure to these compounds.

[0094] In an embodiment, the antibiofilm efficacy of compound 26 in several concentrations was determined through a biofilm prevention assay (compound 26 mixed with inoculum) and a biofilm reduction assay (pre-formed biofilms exposed to compound 26 in solution) using Pseudoalteromonas tunicata (Figure 8). For the biofilm prevention assay, a cell suspension of P. tunicata at an initial concentration of 1 x 10⁸ cells/mL in the marine medium Vaatanen Nine Salt Solution (VNSS) was placed in contact with different concentrations of compound 26 (0, 0.614, 1.535, 3.07, and 6.14 pg/mL) for 24 h in a 12-well polystyrene plate (VWR International, Carnaxide, Portugal). The microplate was incubated at 25 °C and 185 rpm in an orbital shaker with a 25-mm diameter (Agitorb 200ICP, Norconcessus, Ermesinde, Portugal) to allow the formation of biofilm in shear conditions similar to those estimated for a ship in a harbour (Romeu, Alves et al. 2019). For the biofilm reduction assay, 7-day biofilms of P. tunicata were first formed in VNSS in 12-well plates under the previously mentioned orbital shaking conditions and then exposed to the same concentrations of the test compound for 24 h, maintaining the hydrodynamic conditions. At the end of each assay, biofilm cells were removed from the surface and suspended in 0.85 % (w/v) sterile saline solution for counting. Ten microlitres of each cell suspension were placed on a Neubauer chamber (Zuzi, Lisboa, Portugal) and counting was performed under a light microscope with a 10x objective (Nikon Eclipse LV100 microscope, Nikon Corporation, Tokyo, Japan). Three independent biofilm assays, with three technical replicates each, were performed.

[0095] Results of the two assays showed that compound 26 can prevent and reduce pre-formed biofilms of P. tunicata in a concentration-dependent manner (Figure 8).

[0096] In an embodiment, the ecotoxicity effects of compound 26 in Artemia salina (marine crustacean) and Phaeodactylum tricornutum (marine diatom) were assessed. For comparative purposes, the algal inhibition effects on the marine diatom were also tested in this work for the lead compound 1. Artemia salina eggs were hatched in nutrient-enriched seawater for 48 h, at 25 °C. The assay was performed in 96-well microplates containing 15-20 nauplii per well and 200 pL of test solution. Test solutions of compound 26 were prepared in filtered seawater at concentrations of 25 pM and 50 pM. A. salina nauplii were incubated with the test solutions for 48 h, at 25 °C, in the dark and the percentage of mortality was determined. All tests included KzCrzO? as positive control and DMSO as a negative control. In a further embodiment, algal ecotoxicity test (OECD 201) was performed by IK4 TEKNIKER accordingly to the EU hazard assessment of substances and European Ecolabel (ISO 113482). Algal growth inhibition test (MARINE ALGALTOXKIT M™) was used as a standard test to evaluate the marine ecotoxicity of compound 26. The MARINE ALGALTOXKIT M™ contained all the material necessary to perform the growth inhibition tests with the marine diatom P. tricornutum. Water Accommodated Fractions (WAFs) were prepared for compound 26 previously to the exposure to the marine algae. WAFs were prepared by stirring the test substances for at least 24 h in water and subsequently removing the insoluble portions by an appropriate method. The species of alga P. tricornutum, were incubated with the testing leaching samples, for 72 h, in disposable cells of 10 cm path-length. Algal growth or inhibition was registered every 24 h, measuring the optical density (OD) at 670 nm in the spectrophotometer (Jenway 6300), equipped with a holder of 10 cm cells. A dilution series was prepared (32, 18, 10, 5.6, and 3.2 mg/L) and EC50/EL50, as the concentration of the test substance that causes a decrease of 50 % in the growth of the algae was calculated. In order to check the correct execution of the test procedure and the sensitivity of the test, a reference-quality control test was carried out with the reference chemical potassium dichromate (KzCrzC J.ln an embodiment, it was observed that compound 26 caused less than 10 % mortality to A. salina nauplii at concentrations of 25 and 50 pM after 48 h of exposure. The observed lethality was not significantly different (p < 0.01) from the negative control (filtered seawater) and thus it can be concluded that even though its higher potency against the settlement of mussel larvae, compound 26 remains non-toxic to this non-target species, similar to what was previously observed for compound 1 (Almeida, Correia-da-Silva et al. 2017). Considering algal growth inhibition, while compound 1 exhibited toxicity, compound 26 was still classified as non-toxic to this diatom species (Table 2).

Table 2 - Results obtained in the ecotoxicity assay with Phaeodactylum tricornutum performed for compounds 1 and 26.

(The validity criteria for the acceptance of the test results were fulfilled since a concentration of 11.3 mg/L KzCrzO? produced between 20 % and 80 % inhibition after 30 min of exposure.) [0097] In an embodiment, compound 26 was selected to study its viability as an AF agent in commercial marine coatings, based on its AF activity versus toxicity performance against target and non-target organisms. For this purpose, derivative 26 was incorporated in a two component PU-based marine paint as an additive, and which comprises a polyurethane-based resin (Ref. F0032) and a curing agent (Ref. 95580 (Hempel A/S Copenhagen, Denmark). The formulations preparation followed the instructions provided by the paint components suppliers and, in addition, in accordance with previously developed methodologies (Neves, Almeida et al. 2020, Vilas-Boas, Carvalhal et al. 2020). Briefly, for the preparation of the PU-based formulations, compound 26 was first dissolved in /V-methylpyrrolidone in a compound 26/solvent weight ratio of 0.41, giving a solution with a content of compound 26 of 47.76 ± 1.75 wt.%, which was further added and blended into the PU paint components system (base/curing agent ratio = 11/1) in the exact amount to yield the desirable compound 26 content.

[0098] In a further embodiment, this derivative 26 showed good compatibility with a PU-based marine paint and the optimized formulations allowed the incorporation of the derivative at contents as high as 2.04 ± 0.08 wt. % relative to the total weight of the uncured formulation.

[0099] In a further embodiment, the PU-based marine coating comprising compound 26 was then used to coat a 24-well microplate system and the AF activity of the generated 26 compound-based coating was evaluated in laboratory conditions (Figure 9). For the initial anti-macrofouling assessment, M. galloprovincialis plantigrades were collected in Memory beach (N41°13'51.5", W8°43'15.5") at low tide. Competent plantigrades with exploring behaviour were selected in the laboratory and transferred to the coated wells. All the coated wells were filled with filtered and sterilized natural seawater to reduce any interferents. The coating was tested in four replicates (wells) with five plantigrades per well. A negative control (AF agent-free coating system) was included. After 15 and 40 h, the percentage of larval settlement was determined by the presence/absence of efficiently attached byssal threads, produced by each individual, in each condition. [00100] PU-based marine coating containing compound 26 was effective against the settlement of mussel larvae (Figure 9), presenting a larval settlement of only 20 and 10 %, after 15 and 40 h, respectively. A larval settlement of 55 and 30 % was observed in the negative control. The decreased settlement on coatings containing compound 26 represents a good indicator of compound 26 AF potential as an additive for PU-based marine coatings. More relevant, compound 26, behaved better than PU marine coatings containing compound 1, according to the data previously published on compound 1 (Vilas-Boas, Carvalhal et al. 2020).

[00101] In a yet further embodiment, compound 26 may be incorporated in PU- based marine coating formulations at a concentration of 1.00% and 2.00 wt.%. The formulations containing compound 26 (Table 3) were prepared in accordance with the previous procedure to perform the antibiofilm assays and to assess the agent content effect on those.

[00102] In an embodiment, further coating optimization was implemented to prevent the premature release of the AF agent. Compound 26 was conventionally incorporated as an additive in a two-component PU-based marine coating, composed of a base resin F0032 and a curing agent 95580 (Hempel A/S Copenhagen, Denmark. Contents of compound 26 as high as 1.98 ± 0.01 wt.% were obtained in the wet paint formulations. For the preparation of PU-based formulations, compound 26 was first dissolved in /V-methyl pyrrolidone with a compound 26/solvent weight ratio of 0.38, giving a solution with a content of compound 26 of 1.05 ± 0.01 and 1.98 ± 0.01 wt.%, which were further added and blended into the PU components in the exact amounts to yield the desired compound contents in the wet systems (please see Table 3). For the PU-based formulation containing 1.88 ± 0.01 wt.% of compound 26 and the optimized formulation including the trimethylolpropane triaziridine propionate crosslinker (TZA, 99.5%, PZ Global, Barcelona, Spain), a similar preparation methodology was followed, but with a bioactive derivative/solvent weight ratio of 0.35, and a crosslinker content of 1.08 ± 0.48 wt.% in the wet formulation. For all prepared formulations, the base and curing agent weight proportion was 9/1, following the supplier's instructions. The optimized compound 26-based formulations were further used to coat l x l cm² glass slides (coupons) (Vidraria Lousada, Lda, Lousada, Portugal) through a conventional dipping coating procedure. Formulations presented in Table 3 were further used to study the antibiofilm properties of the generated compound 26-based coating system.

Table 3 - PU-based marine coating formulations containing compound 26.

Coating Base/Curing Compound 26 Content TZA Content formulation Agent Ratio (v/v) (wt.%) (wt.%)

Compound 26-PU 1.05 ± 0.01

Compound 26-PU 1.98 ± 0.01

Compound 26- 1.88 ± 0.01 1.08 ± 0.48

TZA/PU

PU: Polyurethane; TZA: trimethylolpropane triaziridine propionate crosslinker.

[00103] In an embodiment, the biofilm formation of Pseudoalteromonas tunicata was evaluated under hydrodynamic conditions. The ability of P. tunicata to colonize the PU-based coatings was monitored for 7 weeks (49 days) using 12-well microplates under the hydrodynamic conditions referred to for the antibiofilm assays. Biofilm development was followed for 49 days because this period corresponds to approximately half of the minimal economically viable interval accepted for the maintenance of underwater systems and hull cleaning. A P. tunicata suspension at a final concentration of 1 x 10⁸ cells/mL was prepared in VNSS medium from the overnight culture. The PU-based surfaces were first fixed to the plate wells using double-sided adhesive tape. Then, 3 mL of culture were added to the wells, and the microplates were incubated for biofilm development. At least three coupons of each surface were removed every 7 days for biofilm analysis. During the incubation period, the culture medium was carefully replaced twice a week. Two independent biofilm formation assays, with at least three technical replicates each, were performed. The removed coupons were gently washed with 3 mL of 0.85 % (w/v) sterile saline solution to remove non-attached microorganisms and analysed regarding the number of biofilm cells. The biofilm architecture was also evaluated through Confocal Laser Scanning Microscopy (CLSM) after 49 days. For total cell counting, coupons were vortexed in 2 mL of saline solution for 3 min to release and homogenize the biofilm cells. Then, 10 pL of each cell suspension were placed on a Neubauer chamber and counting was performed under the light microscope. P. tunicata biofilms were imaged using a Leica TCS SP5 II confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) after 49 days of biofilm formation. Biofilm samples were counterstained with Syto9 (Thermo Fisher Scientific, Waltham, MA, USA), a green cellpermeant nucleic acid marker, for 10 min at room temperature, and then scanned at 40x magnitude with an HCX PL APO CS 40x/1.10 CORR water objective lens at an excitation wavelength of 488 nm (argon laser). The emitted fluorescence was recorded within the range of 460 to 575 nm. A minimum of five stacks of horizontal plane images (512 x 512 pixels, corresponding to 387.5 pm x 387.5 pm) with a z-step of 1 pm were acquired for each biofilm sample. Three-dimensional (3D) projections of biofilm structures were reconstructed from the CLSM acquisitions using the blend mode of the "Easy 3D" function of IMARIS 9.1 software (Bitplane, Zurich, Switzerland). Biofilm biovolume (pm³/pm²) and thickness (pm) were extracted from confocal image series with the plug-in COMSTAT2 run in ImageJ 1.48v software.

[00104] In a further embodiment, Figure 10 shows the biofilm analysis concerning the number of biofilm cells (cells/cm2) of P. tunicata for the three investigated coating formulations. For the 1 wt.% compound 26 PU-based coating, the number of cells increased only from day 14 until day 49. No significant difference was observed between day 7 and day 14, suggesting that a strong antibiofilm activity was exerted in the first 14 days. The antibiofilm effect was also observed for the 2 wt.% compound 26 PU-based coating in the first 21 days, after which the number of biofilm cells started to increase, although never reaching the number of cells observed for the 1 wt.% compound 26 PU-based coating at day 49 (around 1.1 x 1010 cell/cm2).

[00105] Surprisingly, a long-lasting effect was observed for the 2 wt.% compound 26 PU-based coating optimized with the crosslinker (CL), compound 26- TZA/PU, where the number of cells only started to increase from day 28 and the number of biofilm cells was lower (around 4 x 10⁹ cells/cm²) than the number of cells observed for the 2 wt.% compound 26 PU-based coating without CL at day 49 (around 8 x 10⁹ cells/cm²). The structural differences of P. tunicata biofilms formed on the three tested surfaces at the end of the 49-days experiment were evaluated using Confocal Laser Scanning Microscopy. Examples of 3D reconstructions are presented in Figure 11. From this figure, it is possible to observe that a denser and thicker biofilm grew on the 1 wt.% compound 26 PU-based coating (1% Compound 26/PU), confirming the results obtained from the biofilm cell count (Figure 10). On the other hand, biofilms formed on the top of the compound 26-TZA/PU (2% Compound 26/PU/CL) did not cover the entire surface area and only scattered cell aggregates could be seen. Regarding biofilm biovolume, it was significantly lower for the compound 26-TZA/PU surface when compared to 1 and 2 wt.% compound 26 PU- based coatings (p < 0.05, Figure 12A). Accordingly, biofilm thickness was higher for the 1 wt.% compound 26 PU-based coating formulation than for the 2 wt.% compound 26 PU-based coating and the optimized compound 26-TZA/PU (p < 0.05, Figure 12B).

[00106] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[00107] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable.

[00108] The following claims further set out particular embodiments of the disclosure.

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Claims

C L A I M S A compound of general formula (I) or an acceptable salt, an hydrate, a solvate, an enantiomer, a atropisomer, a polymorph or an ester thereof

wherein Ri, Rz, Rs, R4, are independently selected;

Ri is selected from a group consisting of NH2, NHs⁺, amine protecting groups, - N-azole;

R2 is selected from a group consisting of H, alkyl;

R3 is selected from a group consisting of H, alkyl, alkyltriazolefluoroaryl;

R4 is selected from a group consisting of H, alkyl; and wherein n ranges from 1 to 3. The compound according to the previous claim wherein the amine protecting group is selected from a group consisting of carbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine, benzylideneamine, p- toluenesulfonamide. The compound according to any of the previous claims wherein Ri is NHs⁺, or t- butyl carbamate. The compound according to any of the previous claims wherein Fb or F are C1-C6 alkyl or H. The compound according to any of the previous claims wherein Fb or F are methyl or H.

The compound according to any of the previous claims wherein Fb and F are equal. The compound according to any of the previous claims, wherein the salt is a fluoride, chloride, bromide, iodide, acetate, citrate, maleate, or mesylate. The compound according to any of the previous claims wherein the compound is:

Composition comprising the compound described in any of the previous claims 1-

8. Composition according to the previous claim comprising from 0.1 to 10 wt.% of the compound described in any of the previous claims, preferably from 1 to 4 wt.%; more preferably from 1 - 2 wt.%. Composition according to any of the previous claims 9-10 wherein the composition is incorporated in a polymeric formulation. Composition according to any of the previous claims 9-11 wherein the composition is a coating composition. Composition according to any of the previous claims 9-12 wherein the composition is an antifouling paint or varnish composition for protecting underwater surfaces. Composition according to any of the previous claims 9-13 further comprising one of the following additives: dye, polymer, filler, essential oil, stabilizer, surfactant, crosslinker agent, curing agent, biocides, solvent, or mixtures thereof. Composition according to any of the previous claims 9-14 wherein the composition is a solvent-based paint. Composition according to the previous claims 9-15 wherein the composition is a polymeric paint composition, preferably a polyurethane based paint. Use of the compound or composition described in any of the previous claims as an antifouling agent. Use of the compound or composition according to the previous claim as a marine antifouling agent. Use of the compound or composition according to the previous claim as an antimicrofouling agent. Article comprising the compound or composition described in any of the previous claims. Article according to the previous claim wherein the article is a paint, a varnish, a stone, a boat, a surfboard, a net, a buoy, or a medical device. A process for obtaining the compound according to any of the previous claims 1-8 by reacting by scheme 1 or sheme 2 a compound of general formula (II)

wherein Ri, R2, R3, are independently selected;

Ri and R3 are an alkyl; preferably C1-C6 alkyl;

R2 is selected from a group consisting of H, alkyl; preferably C1-C6 alkyl.