XANTHONIC COMPOUNDS AND THEIR USE AS ANTIFOULING AGENTS

Abstract

Synthetic small molecules from an important class of heterocyclic derivatives and homologs and their use as antifouling agents for protection against marine biofouling. The antifouling compounds disclosed herein are environmentally friendly and are able to reduce marine biofouling without inducing toxic effects to target and non-target species. The claimed antifouling compounds also possess ability to be incorporated as antifouling additives in polymeric formulations. Thus, allowing its further application for the preparation of antifouling polymeric matrices, such as coatings, with no hazard effects for the environment.

Claims

1. A compound of formula (I), ##STR00005## salts or esters thereof, for use as antifouling agent, wherein: Y represents oxygen, sulfur, CH.sub.2 or N—H; Z represents C═O, CH.sub.2, CH—OH, C═NOH, C═NOCH.sub.3, NO, NOH, S═O or SO.sub.2; R1, R2, R5, R6, R7 and R8 are independently of each other, selected from hydrogen, hydroxyl, alcoxyl, hydroxyalkyl, alkyl, halogen, alkyl-halogen, trifluoromethyl, acetylene, carboxyl, aldehyde, cyano, nitro, B(OH).sub.2, SO.sub.2NH.sub.2, aryl or heteroaryl substituted by halogen or hydroxyl or methoxyl, amine, aminoalkoxyl, aminoaryl, imine, methylenealkylamine, cycloalkyl, or together an aminoalkyl, alcoxyl, alkylene(di)oxyl group such as pyran or pyran substituted by alkyl, furan or dioxane or dioxane substituted by aryl, or aryl substituted by halogen or hydroxyl or alcoxyl or alkylene(di)oxyl; wherein the compounds are 3,4 substituted; wherein if one of R1 to R8 represents an amine group and/or aminoalkyl, counterions selected from HCO.sub.3.sup.−, CO.sub.3.sup.2−, Cl.sup.−, NH.sub.2C.sub.6H.sub.4SO.sub.3.sup.−, 1-CH.sub.3C.sub.6H.sub.2-3-OH-4(CHCH.sub.3)-6-SO.sub.3.sup.− are coordinated or ionically bound in the amine; wherein at least R3 and R4 are independently selected from hydroxy, optionally substituted C1 to C6 (oxy)alkyl, optionally substituted (oxy)alkylester.

2. The compound for use as antifouling agents according to claim 1, wherein the compound is selected from the group consisting of 3,4-dihydroxy-9H-xanthen-9-one; 3,4-dimethoxy-9H-xanthen-9-one; 3,4-dimethoxy-1-methyl-9H-xanthen-9-one; 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one; 3,4-dimethoxy-1-(((2-morpholinoethyl)amino)methyl)-9H-xanthen-9-one; 1-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one; 1-((5-amino-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one; 3,4-dimethoxy-1-(piperidin-1-ylmethyl)-9H-xanthen-9-one; 1-(dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one; 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde; 1-(hydroxymethyl)-3,4-dimethoxy-9H-xanthen-9-one; dimethyl 2,2′-((9-oxo-9H-xanthene-3,4-diyl)bis(oxy))diacetate and 2,2′-((9-oxo-9H-xanthene-3,4-diyl)bis(oxy))diacetic acid.

3. The compound according to claim 1, for use in the reduction or prevention of marine biofouling.

4. An antifouling formulation comprising the compound of Formula (I) as defined in claim 1.

5. A polymeric matrix composition comprising the compound of Formula (I), as defined in claim 1, in an amount between 1 and 4% wt.

6. The polymeric matrix composition according to claim 5, wherein the matrix is a coating.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0053] For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

[0054] Table 1 illustrates the antifouling effectiveness versus toxicity of compounds towards the anti-settlement of mussel plantigrades. EC.sub.50, minimum concentration that inhibited 50% of larval settlement; LC.sub.50 the median lethal dose; LC.sub.50/EC.sub.50, therapeutic ratio. Note: reference values for EC.sub.50<25 μg.Math.ml.sup.−1 (U.S. Navy program) and therapeutic ratio (LC.sub.50/EC.sub.50) higher than 15.

[0055] Table 2 illustrates the ecotoxicity of compounds towards nauplids of the brine shrimp Artemia salina.

[0056] FIG. 1 illustrates the anti-settlement effect of 3,4dOHx-polyurethane based marine paint coatings containing 2 wt. % (PU-DX1) and 3 wt. % (PU-DX2) of xanthone 3,4dOHx against Mytilus galloprovincialis larvae settlement. 3,4dOHx, 3,4-dihydroxy-9H-xanthen-9-one.

[0057] FIG. 2 illustrates the anti-settlement effect of XA13-polyurethane based marine paint coatings containing 1.7 wt. % of XA13 xanthone against Mytilus galloprovincialis larvae settlement. XA13, 3,4-dimethoxy-1-methyl-9H-xanthen-9-one.

[0058] FIG. 3 illustrates the chemical structure of the xanthonic compounds of the present application.

BEST MODE FOR CARRYING OUT THE INVENTION

[0059] This application relates to synthetic small molecules from an important class of heterocyclic derivatives and homologs and their use as antifouling agents for protection against marine biofouling. More specifically, the present application relates to xanthonic compounds and derivatives and their use as antifouling agents.

[0060] Synthetic 3,4-oxygenated xanthones, aminated and not aminated, were investigated for the first time for their antifouling potential.

[0061] These xanthones were studied in vivo against the settlement of Mytilus galloprovincialis and their mode of action was investigated. Adding to their known pharmacological actions, herein it is disclosed for the first time 3,4-oxygenated xanthonic derivatives, as efficient antifoulants against Mytilus galloprovincialis and as compatible agents for commercial polymeric coatings formulations, allowing their use as additives to provide protective antifouling coatings.

[0062] The xanthones compounds described herein have the structure represented by Formula I:

##STR00002##

their salts or esters, wherein:
Y represents oxygen, sulfur, CH.sub.2 or N—H;
Z represents C═O, CH.sub.2, CH—OH, C═NOH, C═NOCH.sub.3, NO, NOH, S═O or SO.sub.2;
R1, R1, R3, R4, R5, R6, R7 and R8 are independently of each other selected from hydrogen, hydroxyl, alcoxyl, hydroxyalkyl, alkyl, halogen, alkyl-halogen, trifluoromethyl, acetylene, carboxyl, aldehyde, cyano, nitro, B(OH).sub.2, SO.sub.2NH.sub.2, aryl or heteroaryl substituted by halogen or hydroxyl or methoxyl, amine, aminoalkoxyl, aminoaryl, imine, methylenealkylamine, cycloalkyl, or together an aminoalkyl, alcoxyl, alkylene(di)oxyl group such as pyran or pyran substituted by alkyl, furan or dioxane or dioxane substituted by aryl, or aryl substituted by halogen or hydroxyl or alcoxyl or alkylene(di)oxyl;
wherein the compounds are 3,4 substituted; [0063] wherein if one of R1-R8 represents an amine group and/or aminoalkyl this contains counterions such as HCO.sub.3.sup.−, CO.sub.3.sup.2−, Cl.sup.−, NH.sub.2C.sub.6H.sub.4SO.sub.3.sup.−, 1-CH.sub.3C.sub.6H.sub.2-3-OH-4(CHCH.sub.3)-6-SO.sub.3.sup.− which are coordinated or ionically bound in the amine; [0064] wherein at least R3 and R4 are independently selected from hydroxy, optionally substituted C1 to C6 (oxy)alkyl, optionally substituted (oxy)alkylester.

[0065] And the numbers 1, 2, 3, 4, 4a, 5, 6, 7, 8, 8a, 9, 9a, 10 and 10a, represent the numbers of the carbons of Formula I.

[0066] 1. Results

[0067] 1.1 Chemical Synthesis

[0068] According to the previously described procedures, the following 3,4-oxygenated xanthone derivatives were synthesized: 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx); 3,4-dimethoxy-9H-xanthen-9-one (34MX); 3,4-dimethoxy-1-methyl-9H-xanthen-9-one (XA13).

[0069] The pyranoxanthone 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one (XP13) was also obtained.

[0070] As well as the following aminated xanthone derivatives: 3,4-dimethoxy-1-(((2-morpholinoethyl)amino)methyl)-9H-xanthen-9-one (XA15); 1-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one (XA18); 1-((5-amino-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one (XA20); 3,4-dimethoxy-1-(piperidin-1-ylmethyl)-9H-xanthen-9-one (XA22); 1-(dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one (XA14); 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (XA11); 1-(hydroxymethyl)-3,4-dimethoxy-9H-xanthen-9-one (XA12) dimethyl 2,2′-((9-oxo-9H-xanthene-3,4-diyl)bis(oxy))diacetate (D3,4OCH3X) and 2,2′-((9-oxo-9H-xanthene-3,4-diyl)bis(oxy))diacetic acid (D3,4 COOHX).

[0071] The investigated compounds presented purity >95% by HPLC-DAD.

[0072] The following structures some of the embodiments of the xanthone compounds derived from Formula (I).

##STR00003## ##STR00004##

[0073] 1.2 Antifouling Bioactivity

[0074] Dose-response antifouling bioassays confirmed that all compounds showed AF bioactivity towards mussel plantigrade larvae settlement, with levels of effectiveness above the reference values established by the US Navy (EC.sub.50<25 μg.Math.ml.sup.−1) (Table 1).

[0075] Regarding toxicity to mussel larvae, no mortality was found in the range of concentrations tested, as so the LC.sub.50 was considered as higher than the concentration tested for each compound (Table 1).

[0076] 1.3 Non-Target Species Toxicity Assessment

[0077] 1.3.1 Artemia salina Lethality Bioassay

[0078] In the presence of eleven selected xanthones (XP13, 3,4dOHx, D3,4 OCH3X, D3,4 COOHX, XA11, XA12, XA14, DR5, XA15, XA18 and XA22) non-toxic effects were observed, even at 50 μM.

[0079] 1.3.2 Luminescent Vibrio fischeri Assay

[0080] Three compounds selected from the initial AF screening did not exert significant general ecotoxicity, as no inhibition of light radiation emitted was found, either after a contact time of 30 min (3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx) LC50>750 μg/mL; 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one(XP13) LC50>237 μg/mL; 1-((5-amino-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one (XA20) LC50>300 μg/mL).

[0081] 1.3.3 Alga Growth Inhibition Test

[0082] The alga growth inhibition of two compounds was tested using Phaeodactylum tricornutum (marine alga). This type of diatom is among the most common type of phytoplankton.

[0083] Example 1: 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx) EL50 (72 h)>100 μg/mL-non-toxic

[0084] Example 2: 1-((5-amino-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one (XA20) EL50 (72 h)>125 μg/mL-non-toxic.

[0085] 1.3.4 Daphnia acute Immobilization Test

[0086] The swimming capability of Daphnia in contact with different concentrations of one compound was evaluated. The percentage of immobility at 48 h was determined. 1-((5-amino-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one (XA20) EL50 (48 h) >300 μg/mL-non-toxic.

[0087] 1.4 Antifouling Mode of Action

[0088] 1.4.1 Antifouling Targets by Proteomics

[0089] As to provide additional molecular insights on the antifouling properties of 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx) and 12-hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthone (XP13), the proteome of competent M. galloprovincialis plantigrade larvae in response to the antifouling compounds was analyzed by label-free shotgun proteomics. Quantitative protein variations were surveyed employing statistics (non-parametric methods, KW and MW) and hierarchical clustering. Independent statistical tests were performed for each individual compound.

[0090] Exposure of larvae to 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one (XP13) at 6.25 and 25 uM resulted in the significant change of abundances in 14 proteins. Two putative proximal thread matrix proteins (TMPs) were clearly detected in the control group but become undetected in the two groups exposed to the compound 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one(XP13), herein pointing to a significant drop in the expression of these proteins related with the activity of 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one(XP13). TMPs are specifically expressed by bivalve mollusks that adhere to underwater surfaces through the production of byssal threads. Their function is to provide viscoelasticity to the byssal threads (Sagert and Waite, 2009). Hence the inhibition of the two TMPs may be pointed as one of the most critical events underlying 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one (XP13) inhibition of larvae adhesion.

[0091] Finally, 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx) was, between the 2 antifouling compounds, the one that more alterations induced in the Mytilus plantigrade larvae proteome. In total this compound at 12.5 and 50 uM, altered the abundances in 24 proteins, suggesting alterations in a large spectrum of both general and specialized cellular pathways. The results evidence the cellular action of 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx) particularly towards the functions of cytoskeleton, chaperone mediated regulation of protein activity and cell redox status. Also, two putative collagen proteins, protein-2 collagen-like and precollagen P, displayed contrasting patterns of abundance. Mytilus collagen proteins (PreCols) are specific to the byssal threads and comprehend the main constituents of this adhesive structure. Moreover, the byssal thread properties such as resistance to tension and shock absorber are essentially provided by these proteins. The adhesion inhibitory effects of 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx) may well be associated with the change in the abundance of Precols.

[0092] In one embodiment, the compounds of formula (I) are used in antifouling formulations. The content of antifouling compound in the formulation is between 1 and 4% wt. in relation to the total weight of the formulation composition.

[0093] In another embodiment, the compounds of formula (I) are used in antifouling polymeric matrices compositions as antifouling additives. In this embodiment, the compound is added between 1 and 4% wt. in relation to the total weight of the formulation composition.

[0094] In a particular embodiment, the matrix is a two-component polyurethane based marine coating formulation, (Ref. F0032/95580, HEMPEL SA), containing 2 wt. % of incorporated 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx). The 3,4dOHx is prior dissolved in N-methyl-pyrrolidone (NMP) in a 3,4dOHx/NMP ratio=0.35, followed added and blended in the paint components system, the base and curing agent (base/curing agent ratio=9/1).

[0095] 2. Antifouling Activity and Incorporation Effectiveness of Xanthonic Based Marine Coatings

[0096] The antifouling activity of developed xanthonic based marine coatings was assessed in lab conditions against the settlement of the Mytilus galloprovincialis larvae and leaching tests were also performed on those formulations. 3,4-Dihydroxy-9H-xanthen-9-one (3,4dOHx) polyurethane-based formulations are an illustrative example of the potential application of the new xanthones. FIG. 1 shown improved antifouling effects when 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx) is incorporated in a polyurethane (PU) marine paint, with contents as low as 2 wt % a reduction of 55% in the larvae settlement was observed after the first 15 h, which reached a reduction of about 80% after 40 h. In addition, those 3,4-dihydroxy-9H-xanthen-9-one (3,4dOHx) based PU coatings show a low antifoulant leaching from the polymeric matrix, only 0.008% of the incorporated antifoulant leach out after 45 days submerged in artificial seawater. This behavior is associated to the functionality compatibility of this xanthone with the PU matrix.

[0097] A similar behavior, although not so pronounced was observed for the xanthone 3,4-dimethoxy-1-methyl-9H-xanthen-9-one (XA13) with 50% reduction of larval settlement in the first 15 h and 29% reduction after 40 h (FIG. 2).

[0098] The antifoulant optimum content depends on the specific agent bioactivity and compatibility with the paint components, which can be adjusted taken in account the limitations by each particular polymeric system.

[0099] In the foregoing specification, embodiments of the invention have been described with reference to numerous specific details that may vary from implementation to implementation. Thus, the sole and exclusive indicator of what is the invention and is intended by the applicants to be the invention, is the set of claims that issue from this application, in the specific form in which such claims issue, including any subsequent correction. Any definitions expressly set forth herein for terms contained in such claims shall govern the meaning of such terms as used in the claims. Hence, no limitation, element, property, feature, advantage or attribute that is not expressly recited in a claim should limit the scope of such claim in any way. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

[0100] According to the present invention, the compounds herein disclosed can be used as antifouling agents for protection against marine biofouling, allowing their further application for the preparation of antifouling formulations, and antifouling polymeric matrices, such as coatings, with no hazard effects for the environment.