Current anti-fouling methods often involve using highly toxic and environmentally stable compounds such as those comprising copper (as in Cu02) or tin (as in tri-butyltin fluoride, TBTF). These metals remain in the environment and retain their toxicity for many years. Furthermore, these compounds concentrate in plants and animals higher in the food chain, with many adverse effects. For example, organotin has been suspected in failures of certain shellfish crops.

Specifically, spawning failures and shell deformities have been noticed in adult oysters. Such compounds, while effective as anti-fouling agents, are targets of environmental regulations that seek to limit the concentration of heavy metals in the environment. Effective anti-fouling agents with a short and known biological lifetime are therefore of great interest.

An example of an organic anti-fouling agent is dodecyl guanidinium acetate (dodine), a mono-substituted guanidinium salt widely used as a fungicide and bactericide to control scab on hard fruits. It is also used as an industrial biocide and preservative. Dodine shows synergistic anti-foulihg activity in conjunction with other well-known anti-fouling agents such as tributyltin oxide (Evans et al., Stud.

Env. Sci. 28 : 55-64,1986). Dodine, in conjunction with quaternary ammonium salts, is reported by Bidwell et al. (Bidwell et'al., Aquat : Toxicol. 33 : 183-200, 1995) to control the growth of zebra mussels and Asian clams (moluscicidal activity).

Soluble dodine formulations also have been disclosed in U. S. Patent No. 4,816,163, U. S. Patent No. 4,906,385, and Canadian Patent No. 1269927. A method for

preparing an anti-fouling coating from a mixture of dodine and additional biocides has been disclosed in Japanese Patent No. 04225945.

Isothiouronium salts and isothioureas also show biocidal activity. For example, U. S. Patent No. 4,515,813 discloses the lepidoptericidal properties of isothiourea compounds. Fungicidal and bactericidal activity of this class of compounds were also noted. Similarly, the use of pyridyl thiouronium salts as fungicides is disclosed in U. S. Patent No. 3,655,898, and related pyridyl thiouronium N-oxides are useful as wood preservatives as described in Japanese Patent No. 53109903. German Patent No. 2637651 describes the use of S-(p- isopropylbenzyl) thiouronium chloride as one of the biocidal components in a water- based paint formulation. Marine anti-fouling activity by dissolved isothioureas is disclosed in Japanese Patent 05163105.

Most of the previously reported isothiouronium-and guanidinium-containing compounds are significantly soluble in water. Although this is appropriate for applications requiring soluble biocides, successful coating applications in contact with water call for sparingly soluble biocides. Hiroyuki et al. (Hiroyuki et al., Japanese Patent 4225945) describe poorly water soluble salts of dodecylguanidinium with large organic anions such as oleate which may be used as aquatic anti-fouling agents.

Summary In one aspect the disclosure includes compounds of Formula 1 that are useful for anti-fouling coating applications in contact with water or water vapor.

Formula 1 With reference to Formula 1, Y is either nitrogen or sulfur. If Y is nitrogen, the R groups are selected such that at least one R group is an organic radical and the

remaining R'groups are selected from the group consisting of hydrogen and organic radicals. If Y is nitrogen and only one R group is an organic radical, the organic radical has greater than 12 carbon atoms, more particularly from about 16 carbon atoms to about 30 carbon atoms. If Y is sulfur, RD is absent, R is an organic radical, at least one of RA, RB, and RE is an organic radical, and the remaining R groups are selected from the group consisting of hydrogen and organic radicals. In some embodiments, X-is any anion. In other embodiments, X-is any anion except carboxylate of greater than about 10 carbon atoms.

Disclosed compounds of Formula 1 have been made with organic portions intended to lower aqueous solubility and are considered to be sparingly soluble or insoluble. It is believed that the disclosed compounds have aqueous solubilities of less than about 10 mM at room temperature and function as anti-fouling agents in coating applications by virtue of their anion exchange properties rather than their detergent properties.

In another aspect, the disclosure includes compounds of Formula 2. The compounds of Formula 2 are conjugate bases of the compounds described by Formula 1 and hence even less water soluble.

Formula 2 With reference to Formula 2, Y is either nitrogen or sulfur. If Y is nitrogen, the R groups are selected such that at least one R group is an organic radical and the remaining R groups are selected from the group consisting of hydrogen and organic radicals. If Y is nitrogen and only one R group is an organic radical, the organic radical has greater than 12 carbon atoms, more particularly from about 16 carbon atoms to about 30 carbon atoms. If Y is sulfur, RD is absent, Rc is an organic radical, at least one of RA, RB, and RE is an organic radical, and the remaining R groups are selected from the group consisting of hydrogen and organic radicals. In

some embodiments, X'is any anion. In other embodiments, X-is any anion except carboxylate of greater than about 10 carbon atoms.

As used herein the term organic radical includes alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cyclic alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic groups.

In another aspect, the disclosure provides poly-substituted isothiouronium salts of Formula 3.

Formula 3 With respect to Formula 3, Rl, R2, R3 and R4 groups are selected such that R4 and at least one other R group are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cyclic alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic. The remaining R groups are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cyclic alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic. X'may be any monovalent or polyvalent anion.

For example, X-may be an anion selected from the group consisting of halide (fluoride, chloride, bromide, iodide), nitrate, nitrite, bicarbonate, carbonate, carboxylate (e. g. acetate), dicarboxylate and their mono-protonated analogs (e. g. oxalate and hydrogen oxalate), tricarboxylate and their mono-and di-protonated analogs (e. g. citrate, hydrogen citrate, and dihydrogen citrate), sulfonate (e. g.

aliphatic sulfonate, including alkyl sulfonate, such as methylsulfonate, and aryl sulfonate, such as toluenesulfonate), sulfate, sulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, tetrafluoroborate, hexafluoroborate, hexafluorophosphate, tetraphenylborate, borate, and mixtures thereof.

In another aspect, the disclosure provides poly-substituted guanidinium salts of Formula 4 Formula 4 With respect to Formula 4, R1, R2, R3, R5 and 1t6 groups are selected such that at least two of the R groups are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cyclic alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic. The remaining R groups are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cyclic alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic. X-may be any monovalent or polyvalent anion. For example, X may be chosen from the group consisting of halide (fluoride, chloride, bromide, iodide), nitrate, nitrite, bicarbonate, carbonate, carboxylate (e. g. acetate), dicarboxylate and their mono- protonated analogs (e. g. oxalate and hydrogen oxalate), tricarboxylate and their mono-and di-protonated analogs (e. g. citrate, hydrogen citrate, and dihydrogen citrate), sulfonate (e. g. aliphatic sulfonate, including alkyl sulfonate, such as methylsulfonate, and aryl sulfonate, such as toluenesulfonate), sulfate, sulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, tetrafluoroborate,

hexafluoroborate, hexafluorophosphate, and tetraphenylborate, borate, and mixtures thereof.

Isothiouronium ions and neutral isothioureas may, in certain disclosed embodiments, be interconverted by changes in pH, since such compounds are related as acid and conjugate base. Therefore, the disclosure also includes neutral isothioureas of Formula 5.

Formula 5 With respect to Formula 5, Rl, R2, R3 and R4 groups are selected such that R4 and at least one other R group are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cycolalkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic. The remaining R groups are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cyclic alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic.

Similarly, guanidinium ions and neutral guanidines are related as acid and conjugate base. Therefore, the disclosure also includes compounds of Formula 6.

Formula 6

With respect to Formula 6, Rl, R2, R3, R5 and R6 groups are selected such that at least two of the R groups are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cyclic alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic. The remaining R groups are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted cyclic alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, substituted aliphatic heterocyclic, and substituted aromatic heterocyclic.

In some embodiments, the disclosure provides an anti-fouling agent (also known as a toxicant or biocide in the art of anti-fouling coatings) comprising at least one of the aforementioned compounds.

Other embodiments include an anti-fouling coating composition comprising at least one of the aforementioned compounds. In particular disclosed embodiments, the coating composition comprises an effective anti-fouling amount of at least one of the aforementioned compounds and a binder. In other more particular embodiments the coating composition comprises an effective anti-fouling amount of at least one of the aforementioned compounds, a binder, and one or more additional inert or active components, such as materials selected from the group consisting of binders, pigments, thickeners, extenders, hydrolysis regulators, inert diluents, plasticizers, additional anti-fouling agents, and combinations thereof.

A method of protecting a surface from fouling by applying an effective anti- fouling amount of at least one of the aforementioned compounds also is disclosed.

In particular embodiments, the effective anti-fouling amount is applied as an anti- fouling coating composition comprising such compounds.

Detailed Description- Without restricting the disclosed embodiments to one theory of operation, it is currently believed that the biocidal and anti-fouling activity exhibited by the compounds of the disclosure may be in part due to their anion exchange properties.

Undisclosed results for guanidinium salts used in ion exchange membranes for dissolved gas sensors showed that they are effective agents for the exchange of hydroxide ions across membranes. In the context of biocidal activity, an anion exchanger might serve to disrupt the normal ionic and pH balance across a cell membrane that would prove to be fatal or disruptive for living organisms.

Prior art discussions of the mode of action of biocidal formulations containing isothiouronium or guanidinium salts focussed on their detergent capabilities, a property that may be diminished in some of the disclosed compounds due to their more symmetric hydrophobic substitution patterns. Thus, the surprising activity of the disclosed compounds may in fact be explained by the role that anion exchange capacity plays in biocidal activity rather than their detergent capabilities.

In addition to the biocidal and anti-fouling activity disclosed below, the compounds of the disclosure possess at least two additional properties of significant utility. First, they are colorless; i. e. they do not absorb significant amounts of visible light. and therefore may be used to inhibit fouling on windows exposed to humid or aqueous environments. Second, they degrade easily in a marine environment to produce benign by-products. Thus a buildup of these compounds in the environment may be avoided.

The foregoing and additional aspects and advantages of the compounds, compositions, and methods of the present disclosure are further illustrated by the following discussion and examples. The examples are provided to assist understanding of these aspects and advantages and are not meant to limit the scope of the disclosed embodiments.

General Synthesis Poly-substituted isothiouronium salts of Formula 3, and poly-substituted guanidinium salts of Formula 4 may be prepared according to the following general procedures.

Procedure A Procedure B Procedure C Procedure D Procedure A can be used to produce monosubstituted guanidinium salts or N, N-disubstituted guanidinium salts, depending on the starting amine. The thiouronium salt used in procedure A may be any thiouronium salt, for example, aliphatic or aryl thiouronium salts, such as S-methyl thiouronium iodide, S-ethyl

thiouronium iodide, S-benzyl thiouronium bromide, and S-allyl thiouronium bromide. Reactive organic halides (RX) aid production of the initial thiouronium salt reagent. Volatile thiol (RSH) products are conveniently separated from the guanidinium salt product. Procedure B can produce N, N'-disubstituted thioureas or N, N, N' trisubstituted thioureas, depending on the starting amine. The first step of procedures C and D can produce N, N, S-trisubstituted isothiouronium salts, N, N', S- trisubstituted isothiouronium salts or N, N, N', S-tetrasubstituted isothiouronium salts, depending on the starting thiourea. Direct reaction of isothiouronium salts with ammonia (procedure C) gives either N, N'-disubstituted guanidinium salts or N, N, N' trisubstituted guanidinium salts. Alternatively, reaction of isothiouronium salts with primary or secondary amines (procedure D) can produce N, N', N"-trisubstituted guanidinium salts, N, N, N', N"-tetrasubstituted guanidinium salts, or N, N, N', N', N"- pentasubstituted guanidinium salts, depending on the starting isothiouronium salt and starting amine.

In solution, guanidinium and isothiouronium salts produced by the above reactions, will exist in equilibrium with their neutral conjugate bases. The neutral conjugate bases may be produced by reaction with a base that has a pKb that is smaller than the pKb of the neutral guanidine or isothiourea desired. For example, common bases such as sodium hydroxide, potassium hydroxide, and sodium carbonate may be used. In particular embodiments, isothiouronium and guanidinium salts may be converted to neutral isothioureas and guanidines by treatment with sodium carbonate in ether. In other particular embodiments, isothiouronium and guanidinium salts may be converted to neutral isothioureas and guanidines by passing a methanol solution of the salt through a strongly basic anion exchange resin such as a quaternary ammonium ion exchange resin.

The substituent R groups Rl, R2, R3, R4, R5, R6 in Formulas 1,2,3,4,5, and 6 may be varied by using starting amines and isothiocyanates with those groups in the procedures outlined above. R groups chosen from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cyclic alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aliphatic heterocyclic, aromatic heterocyclic, and substituted aliphatic

heterocyclic, and aromatic heterocyclic groups may have between 1 and about 30 total carbon atoms. In the case of unsubstituted and substituted alkenyl and alkynyl groups, such R groups may have between 2 and about 30 carbons. In the case of cycloalkyl groups and substituted cycloalkyl groups, such R groups may have between 3 and about 30 carbons. Cyclic alkenyl and substituted cyclic alkenyl groups may have between 4 and about 30 carbons. Unsubstituted and substituted aryl groups may have between 6 and about 30 carbon atoms. Heterocyclic groups, both aliphatic and aromatic, may have between 3 and about 30 total carbon atoms.

Particular examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and straight chain and branched alkyl groups having from 6 to 30 carbons, with disclosed embodiments having from about 10 to about 20 carbons.

Particular examples of cycloaklyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

Particular examples of alkenyl groups include vinyl, allyl, butenyl, and pentenyl.

Particular examples of cyclic alkenyl groups include cyclopentenyl, cyclohexenyl, and cyclopentadienyl.

Particular examples of alkynyl groups include ethynyl, propynl, butynyl, and pentynyl.

Particular examples of aryl groups include phenyl and napthyl.

Aliphatic and aromatic heterocyclic groups are radicals derived, respectively, from non-aromatic and aromatic cyclic compounds having in their ring system one or more atoms selected from the group consisting of nitrogen, sulfur, or oxygen atoms and combinations thereof. Particular examples include diazolyl, 2-furoyl, 3- furoyl, furyl, furfuryl, imidazolidyl, imidazolyl, indolyl, isoindolyl, isoxazolyl, isoquinolyl, morpholinyl, oxazolyl, oxadiazolyl, piperidyl, pseudoindolyl, pyranyl, pyrazinyl, pyrazolidyl, pyrazolyl, pyridyl, pyrimidyl, pyrolidyl, pyrrolyl, pyrryl, quinazolyl, quinolyl, quinonyl, quinoxalyl, tetrazolyl, thenoyl, thenyl, thiazolidyl, thiazolyl, thienyl, triazinyl, and triazolyl.

Substituted alkyl, alkenyl, alkynyl, aryl, aliphatic heterocyclic and aromatic heterocyclic groups include groups where at least one hydrogen of the unsubstituted analog is replaced by a halogen, an aliphatic group of 1 to 10 carbons (for example, alkyl, alkenyl, alkynyl), a thiol group, or a hydroxyl group.

The counterion (X~) produced in the syntheses as outlined above is typically halide, such as iodide. This halide ion may subsequently be exchanged for other monovalent and polyvalent anions by ion exchange. For example an iodide ion may be exchanged for other halide ions, such as fluoride, chloride, and bromide. The halide ion may also be exchanged for other anions such as nitrate, nitrite, bicarbonate, carbonate, carboxylate (e. g. acetate), dicarboxylate and their mono- protonated analogs (e. g. oxalate and hydrogen oxalate), tricarboxylate and their mono-and di-protonated analogs (e. g. citrate, hydrogen citrate, and dihydrogen citrate), sulfonate (e. g. toluenesulfonate), sulfate, sulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, tetrafluoroborate, hexafluoroborate, hexafluorophosphate, and tetraphenylborate, borate, and mixtures thereof.

Example 1-Synthesis and Characterization of Alkyl-substituted Isothiouronium Salts, Isothioureas, and Guanidinium Salts A total of fifteen alkyl-substituted compounds were prepared according to the methods outlined above. These compounds included three tri-substituted isothiouronium salts having Formula 3, three di-substituted thioureas having Formula 5, and a total of nine mono-, di-, and tri-substituted guanidinium salts having Formula 4. Specific reaction conditions and reagents used to make the series of compounds are given below. Each of these methods produced highly pure compounds as desired without recourse to purification procedures, such as chromatographic separation. However, samples of each compound were further purified by chromatography on silica. The purified materials all showed UV cutoff values below 300 nm, and showed molar absorptivities of less than one at 300 nm for all compounds. Nuclear magnetic resonance (NMR), mass spectrometry (MS), and infrared spectrometry (IR) were used to further characterize the compounds.

The following specific procedures, given for compounds bearing alkyl substituents, are illustrative of the methods that may be used to prepare compounds according to the disclosure.

Procedure A: ( ! ! CAUTION!!: This procedure evolves methyl mercaptan. Use a hood.

Avoid exposure.) An alkyl amine (1 eq.) and solid S-methyl isothiouronium iodide (2 eq.) were suspended in absolute ethanol (5-8mL/g salt). The mixture was stirred at reflux under a reflux condenser. The evolution of methyl mercaptan was followed using moistened lead acetate test paper. The reaction was usually complete in 6 hours, but the reflux was continued overnight. The mixture was evaporated to a solid and redissolved in water. The water was extracted on a continuous extractor overnight using chloroform, the extracts were dried over magnesium sulfate, filtered and evaporated to yield the iodide salt of the product. The iodide was converted to the chloride using Amberlite IRA400 resin in methanol.

Spectroscopic data for compounds prepared by procedure A: N-decylguanidinium chloride (Formula 4 where Ri = CloH21, R2 = R3 = Rs = R6 = H, X~ = C1-) : lH NMR (CDC13, 8) : 0.95 (br. t., 3H), 1.54 (br. s., 14H), 1.60 (br. m., 2H), 3.10 (t., 2H), 4.90 (br. s., >5H) ; 13C NMR (CDC13, 8) : 14.2,22.7,26.7, 28.5,29.3 (m), 31.9,42.5,156.6; MS (+LSIMS, mNBA) : 200.2 (M-C1).

N-tetradecylguanidinium chloride (Formula 4 where Ri = Cl4H29, R2 = Rs = R5 = R6 = H, X = CU) : lH NMR (CDC13, 6) : 0.95 (br. t., 3H), 1.5 (br. s., 22H), 1.6 (br. m., 2H), 3.10 (t., 2H), 4.9 (br. s., >5H) ; MS (+LSIMS, mNBA) : 256.2 (M-Cl).

N-octadecylguanidinium chloride (Formula 4 where Ri = C18H37, R2 = R3 = R5 = R6 = H, X = I-) : lH NMR (DMSO-d6, 8) : 0.95 (br. t., 3H), 1.54 (br. s., 30H), 1. 60 (br. m., 2H), 3.10 (t., 2H), 4.90 (br. s., >5H) ; MS (+LSIMS, mNBA) : 312.2 (M -I).

Procedure B: ( ! ! CAUTION!!: isothiocyanates are typically lachrymators. Use a hood.

Avoid exposure.) An alkyl amine (1 eq.) and an alkyl isothiocyanate (1 eq.) were dissolved in toluene (5mL/g amine). The mixture was stirred at reflux for 3-5 hr.

The product precipitated in some cases. The mixture was concentrated under reduced pressure, cooled, and filtered. The precipitate was washed with pentane and air-dried. The product is sufficiently pure for the subsequent reaction with either procedure C or D.

Spectroscopic data for the compounds prepared by procedure B: N-butyl-N'-decylisothiourea (product of procedure B where Ri = CloH21, R2 = H, R3 = C4H9) :'H NMR (CDC13, 8) : 0.95 (m., 6H), 1.3 (br. s., 16H), 1.60 (m., 4H), 3.4 (br. s., 4H), 5.7 (br. s., 2H); 13C NMR (CDC13, 8) : 13.7,14.1,20.2,22.7, 26.9,29.5 (m.), 31.0,31.9,44.0 (br.), 139.5; MS (+LSIMS, mNBA) : 273.2 (M+H).

N-butyl-N'-tetradecylisothiourea (product of procedure B where Ri = C14H29, R2 = H, R3 = C4H9) : 1H NMR (CDC13,8): 0.95 (m., 6H), 1.3 (br. s., 24H), 1.60 (m., 4H), 3.4 (br. s., 4H), 5.7 (br. s., 2H); 13C NMR (CDC13, 8) : 13.7,14.1, 20.2,22.7,26.9,29. 5 (m.), 31. 0, 31. 9,44.0 (br.), 139. 5: MS (+LSIMS, mNBA) : 329.2 (M+H).

N-butyl-N'-octadecylisothiourea (product of procedure B where Ri = C18H37, R2 = H, R3 = C4H9) : 1H NMR (CDCl3, 8) : 0.95 (m., 6H), 1. 3 (br. s., 32H), 1.60 (m, 4H), 3.4 (br. s., 4H), 5.7 (br. s., 2H); 13C NMR (CDC13, 8) : 13. 7, 14.1,20.2,22.7, 26.9,29.5 (m.), 31.0,31.9,44.0 (br.), 139. 5 ; MS (+LSIMS, mNBA) : 385. 2 (M+H).

Procedure C (To isothiouronium salt stage): ( ! ! CAUTION!!: This procedure evolves methyl mercaptan. Use a hood.

Avoid exposure.) A substituted thiourea (1 eq.) was suspended in absolute ethanol

(5-lOmL/g, thiourea may not dissolve) and iodomethane (3 eq.) was added. The mixture was sealed in a hydrogenation bottle, heated to about 80°C, and stirred. The hydrogen pressure was typically below about 30 psi. After cooling, the vessel was opened and the unreacted iodomethane and solvent were removed by evaporation.

The product at this stage is an isothiouronium salt of Formula 3 and may be worked up as is or treated with ammonia to form the guanidinium salt as described below.

The iodide was converted to the chloride using Amberlite IRA400 resin in methanol.

Spectroscopic data for compounds prepared by procedure C (to isothiouronium salt stage): N-butyl-N'-decyl-S-methylisothiouronium chloride (Formula 3 where Rl = CioH2i, R2 = H, R3 = C4H9, R4 = CH3, X~ = C1-) : lH NMR (CDC13, S) : 0.9 and 0. 95 (2 br. t., 6H), 1.2 (br. s., 16H), 1.6 (br. m., 4H), 2.8 (br. s. 3H), 3.75 (m., 4H), 7.8 (br. 2H) ; 13C NMR (CDC13, 5) : 13.7,14.1,19.8,22.6,27.0,29.3 (m.), 31.. 8,44.7, 45.0,50.1,166.5; MS (+LSIMS, mNBA) : 287.4 (M-C1).

N-butyl-S-methyl-N'-tetradecylisothiouronium chloride (Formula 3 where Rl = Cl4H2o, R2 = H, R3 = C4H9, R4 = CH3, X~ = I-) : lH NMR (CDC13, 8) : 0.9 and 0.95 (2 br. t., 6H), 1.2 (br. s., 24H), 1.6 (br. m., 4H), 2.8 (br. s. 3H), 3.75 (m., 4H), 7.85 (br. 1H), 8. 4 (br., 1H) ; 13C NMR (CDC13,8): 13.7,14.1,19.7,22.9,27.1,29.4, (m.), 31.8,44.7,45.1,50.3,166.5; MS (+LSIMS, mNBA) : 343.4 (M-I).

N-butyl-S-methyl-N'-octadecylisothiouronium chloride (Formula 3 where R = CisH37, R2 = H, R3 = C4H9, R4 = CH3, X = I-) : lH NMR (CDC13, oa) : 0.9 and 0.95 (2 br. t., 6H), 1.2 (br. s., 32H), 1.6 (br. m., 4H), 2.8 (br. s. 3H), 3.75 (m., 4H), 8.0 (br. 2H); 13C NMR (CDC13, 8) : 13.7,14.1,19.7,22.6,27.1,29.3 (m.), 31.8,44.7, 44.8,50.3,166.5; MS (+LSIMS, mNBA) : 399.4 (M-I).

Procedure C (to guanidinium salt stage): The isothiouronium iodide salts initially prepared by Procedure C above were dissolved in absolute ethanol (5mL/g) and ammonia was added via a bubbler at a rate to allow dissolution. Ammonia addition was continued until a large excess was assured. The mixture was again sealed and heated at 80°C for 24 hours. After cooling the vessel was opened ( ! ! CAUTION!!: use a fume hood) and reheated to drive off the methyl mercaptan. The mixture was then evaporated to a thick oil.

The oil was dissolved in water, extracted with methylene chloride, the extracts were dried over magnesium sulfate, filtered and evaporated to yield the iodide salt of the product. The iodide was converted to the chloride using Amberlite IRA400 resin in methanol.

Spectroscopic data for compounds prepared by procedure C (to guanidinium salt stage): N-butyl-N'-decylguanidinium chloride (Formula 4 where Ri = CloH2i, R3 = C4H9, R2 = R5 = R6 = H, X~ = C1-) : lH NMR (CDC13, b) : 0.9 and 0.95 (2 br. t., 6H), 1.2 and 1.4 (br. s. + m., 16H), 1.6 (br. m., 4H), 3.15 (br. m., 4H), 6.4-7.0 (br., 4H); 13C NMR (CDC13, 8) : 13.7,14.1,20.0,22.7,26.8,29.4 (m.), 31.9,42.2,42.5,155.8 ; MS (+LSIMS, mNBA) : 256.4 (M-Cl).

N-butyl-N'-tetradecylguanidinium chloride (Formula 4 where Ri = C14H29, R3 = C4H9, R2 = R5 = R6 = H, X = Cl-) : lH NMR (CDCl3, 8) : 0.9 and 0.95 (2 br. t., 6H), 1.2 and 1.4 (br. s. + m., 24H), 1.6 (br. m., 4H), 3.15 (br. m., 4H), 6.4-7.0 (br., 4H); 13C NMR (CDC13, 8) : 13.7,14.1,19.9,22.7,26.9,29.4 (m.), 31.9,41.8,42.1, 156.2; MS (+LSIMS, mNBA) : 312.5 (M-Cl).

N-butyl-N'-octadecylguanidinium chloride (Formula 4 where Ri = C18H37, R3 = C4H9, R2 = R5 = R6 = H, X = Cl-) : lH NMR (CDC13,8): 0.9 and 0.95 (2 br. t., 6H), 1.2 and 1.4 (br. s. + m., 32H), 1.6 (br. m., 4H), 3.15 (br. m., 4H), 6.4-7.0 (br., 4H); MS (+LSIMS, mNBA) : 368.6 (M-Cl).

Procedure D: The procedure was identical to procedure C to the guanidinium salt stage with the exception that an alkyl amine (2 eq.) was used in place of ammonia.

Spectroscopic data for compounds prepared by procedure D: N, N'-dibutyl-N"-decylguanidinium chloride (Formula 4 where Ri = CloH2l, R3= Rs = C4H9, R2 = R6 = H, X = C1-) : lH NMR (CDC13, 8) : 0.95 (m., 9H), 1.2-1.4 (m., 18H), 1.6 (m., 6H), 3.25 (br. m., 6H), 7.0-7.2 (br., 3H) ; 13C NMR (CDC13, 8) : 13.7,13.8,14.1,19.9,20.0,22.7,26.8,29.4 (m.), 31.8,31.9,42.3,42.6,155.4; MS (+LSIMS, mNBA) : 312.2 (M-C1).

N, N'-dibutyl-N"-tetradecylguanidinium chloride (Formula 4 where Rl = C14H29, R3= R5 = C4H9, Ra = R6 = H, X'=Cl'):'H NMR (CDC13, 8) : 0.95 (m., 9H), 1.2-1.4 (m., 26H), 1.6 (m., 6H), 3.25 (br. m., 6H), 7.0-7.2 (br., 3H); 13C NMR (CDC13, 8) : 13.7,14.1,19.9,22.7,26.8,29.4 (m.), 31.8,31.9,42.5,42.8,155.7; MS (+LSIMS, mNBA) : 368.6 (M-C1).

N, N'-dibutyl-N"-octadecylguanidinium chloride (Formula 4 where Rl = Ci8H37, Rs= R5 = C4H9, Rz = Ré = H, X'=I'):H NMR (CDC13, 8) : 0.95 (m., 9H), 1.2-1.4 (m., 34H), 1.6 (m., 6H), 3. 25 (br. m., 6H), 7.0-7.2 (br., 3H); 13C NMR (CDC13, b) : 13.7,14.1,19.9,22.7,26.8,29.4 (m.), 31.8,31.9,42.5,42.8,154.6; MS (+LSIMS, mNBA) : 324.7 (M-I).

Example 2-Assessment of Marine Anti-fouling Activity This example concerns the behavior of the compounds of the disclosure when added to paint formulations that did not contain added biocides. The compounds inhibited the growth of marine organisms on treated surfaces subjected to prolonged immersion in open seawater.

An experiment patterned after a standard anti-fouling assessment method (ASTM D3623-78a, American Society for Testing and Materials, West

Conshohocken, PA) was designed to follow the onset and development of algal growth on painted panels held approximately lm below the surface in open seawater. The apparatus consisted of a moored floating superstructure with a set of test panels suspended below it. The superstructure allowed the test panels to be lifted from the water periodically to assess the extent of growth and to photograph the panels. The apparatus was designed to hold 90 test panels, each 10 cm square.

Nine of the compounds prepared as described in Example 1, at two different nominal dose levels (5 and 10 wt%) in three different topside marine paints (9 x 2 x 3 = 54 primary samples) were compared. None of the paints contained any commercial anti-fouling agent. A total of 12 control samples were included: 6 of which were left intact throughout the one-year experiment and 6 of which were used to provide samples to determine the type of organisms populating the surface fouling layer. The remaining 24 test panels were assigned to randomized replicates of the primary samples, in sets of 8 for each paint type. The locations of the controls were fixed on the six arrays and the remaining 78 test panels were randomly assigned to the other locations.

Lexan test panels (in place of the primed metal panels called for in the ASTM method) were sandblasted to provide a surface for paint adhesion, cleaned in methanol, and then in trifluorethanol immediately prior to painting. Paint samples were prepared from a weighed amount of the test compound and a known volume of paint using the measured paint density to arrive at the nominal 5 and 10 wt% dose levels. In most cases, the compounds were dissolved in a few mL of methylene chloride before the paint was added. The paint samples were mixed by hand until homogeneous to the eye. A measured volume of the paint sample was spread on the cleaned test panel with a silk-screen tool and a jig designed to form a 250 um paint layer. The painted test panels were then glued in place on the array and allowed to air dry for 72 hours. After painting, the sole identifier for the compound and the topside paint used in each formulation was its position on the array. Given that the six arrays were virtually indistinguishable after drying, the specific location of any particular compound was essentially hidden from the subsequent observers.

The experiment was initiated in the summer. Qualitatively, the panels remained clean for the first two weeks, then rapidly fouled over the next two weeks.

By the end of six-week period, the late-summer die-off of marine flora was evident from the amount of plant debris in the water column and the exposure of some previously fouled surfaces on the test panels. The main fouling observed was filamentous algae that hung from the frame of the apparatus, from the clean sections between the test panels, and from some fouled panels.

The extent of fouling was assessed and scored by two independent observers.

Statistical controls establish excellent agreement between the observers. The observers scored the control panels as"heavily"fouled after a six-week exposure.

At the same time, a total of 8 test panels corresponding to 6 compound-dose-paint formulations showed significantly less growth than the controls. Some test panels remained completely free of algal growth after the six-week exposure.

Formulations containing N-butyl-N'-decyl-S-methylisothiouronium chloride (Formula 3 where Rl= CIOH21, R3= C4H9, R2= H, R4= CH3, X = C1-) showed virtually no growth over the first six weeks of the experiment in the three different paint formulations. In two of the paint formulations, growth on panels containing N-butyl-N'-tetradecylguanidinium chloride (Formula 4 where Ri = C14H29, R3 = C4H9, R2 Rs-R5= H, X = C1) was inhibited relative to controls during the initial growth period, but increased after five weeks to levels that were less fouled but not statistically significant relative to controls. These data establish that these compounds inhibit initial growth on the surfaces.

After a 9-month exposure all control panels and untreated surfaces were heavily fouled with brown and green algae, and barnacles had set in many places.

Several other organisms inhabited regions of the dense algal mat around and on the painted panels. Several test panels were significantly less fouled than control surfaces with a substantial portion of the surface (>90% in some cases) free of attached algae and barnacles. All paint formulations containing N-butyl-N'-decyl-S- methylisothiouronium chloride (Formula 3 where Ri = CloH2i, R3 = C4H9, Ruz = H, R4= CH3, X = C1-) showed clear dose dependent anti-fouling activity. The majority of the paint formulations containing N-butyl-N'-octadecylguanidinium chloride

(Formula 4 where R, = Cl8H37, R3 = C4H9, R2 = R5 = R6 = H, X'= Cl') and N-butyl- N'-decylguanidinium chloride (Formula 4 where Ri = CloH21, R3 = Rs = C4H9, R2 = R6 = H, X-= Cl-) also showed dose-dependent anti-fouling activity. These data establish that these compounds inhibit marine growth on treated surfaces, both during the initial colonization phase, and over the longer term.

After one year of exposure, the panels were removed and scored in a manner similar to that found in the ASTM method to provide a quantitative fouling index.

The results of the scoring are found in Table 2 below, where a score of 100 corresponds to no fouling and a score of 0 corresponds to a panel being totally fouled.

These results show that after one year of stationary exposure, panels treated with N-butyl-N'-decyl-S-methylisothiouronium chloride and N-butyl-N'- octadecylguanidinium chloride exhibited statistically significant, dose-dependent reductions in fouling compared to the control panels for more than one base paint formulation. The compounds N, N'-dibutyl-N"-decylguanidinium chloride, N, N'- dibutyl-N"-tetradecylguanidinium chloride, and N, N'-dibutyl-N"- octadecylguanidinium chloride show statistically significant dose-dependent reductions in fouling compared to the control panels in one of the base paint formulations. Better results might be expected for these compounds in a self- polishing coating formulation under typical conditions where there is sustained relative motion of water over the surfaces.

Table 2 Compound Weight % PacP Urethane ICI N-decylguanidiniumchloride 6 5 68* 0 N-decylguanidiniumchloride 11 0 0 36 N, N'-dibutyl-N"-decylguanidinium chloride 6 0 43 20 N, N'-dibutyl-N"-decylguanidinium chloride 11 39* 86 6* N-butyl-N'-decyl-S-methylisothiouroniumchloride 6 0 88* 40 N-butyl-N'-decyl-S-methylisothiouroniumchloride 11 53 94 74 N-tetradecylguanidiniumchloride 74 71 41* N-tetradecylguanidiniumchloride 11 0 0 2* N-butyl-N'-tetradecylguanidiniumchloride 6 42* 0 55 N-butyl-N'-tetradecylguanidinium chloride11"0 17* 29* N, N'-dibutyl-N"-tetradecylguanidinium chloride 5 24 69 24* N, N'-dibutyl-N"-tetradecylguanidinium chloride 10 0 86 0 N-octadecylguanidiniumchloride 5 0 55 0 N-octadecylguanidiniumchloride 10 68 16 10 N-butyl-N'-octadecylguanidinium chloride 5 69 56 0 N-butyl-N'-octadecylguanidiniumchloride 10 78 19 88 N, N'-dibutyl-N"-octadecylguanidinium chloride 5 28 19* 0 N, N'-dibutyl-N"-octadecylguanidinium chloride 10 L 55 77* 50

Bold indicates that the test panel was significantly less fouled than the control (90% confidence interval) AND there was a significant dose dependence of the effect.

*Indicates averaged replicates.

PacP-"Pacific Sailor", Consolidated Coatings, Delta, B. C. Canada ICI-a marine enamel from ICI Paints, Concord, Ont., Canada.

Urethane-Interlux Brightside single part polyurethane, Vancouver, B. C., Canada As used herein, an effective anti-fouling amount of an anti-fouling agent is an amount of the agent that is equal to or greater than the minimum amount or concentration needed to exhibit a statistically significant reduction in the amount of fouling of a surface as judged by ASTM D3623-78a, the method outlined above in this Example, or any other method which provides an anti-fouling index. Other examples of fouling tests that may be utilized to provide an anti-fouling index include ASTM method D4939-89,"Standard Method for Subjecting Antifouling Coating to Biofouling and Fluid Shear Forces in Natural Seawater and ASTM method D5479-94,"Standard Practice for Testing Biofouling Resistance of Marine Coatings Partially Immersed in Seawater.

Example 3-Degradation of Isothiouronium Salts in Seawater Stability in seawater over a period of time was examined to determine the rate of degradation. Seawater samples (20 L) containing N-butyl-N'-decyl-S- methylisothiouronium chloride (Formula 3 where Ri = Cloth21, R2 = H, R3 = C4H9, R4 = CH3, X'= Cl-) at an initial concentration of 100 nM were held at 11°C in an east-facing window. Air was bubbled through the samples for 2 hours each day to maintain saturation. At intervals over 5 days, 100 mL samples of seawater were withdrawn and analyzed by electrospray mass spectrometry. A steady decline in the concentration of the compound was observed with an apparent half-life of 80 hours under the experimental conditions. This experiment establishes that the compound is likely to degrade in the environment. The product of the degradation is initially the corresponding urea that is then further degraded in the seawater.

Example 4-Coating Compositions The disclosed poly-substituted isothiouronium salts, guanidinium salts, neutral isothioureas, and neutral guanidines may be used as anti-fouling agents (also known in the art as biocides or toxicants) in coatings that are applied to surfaces.

Such anti-fouling coatings inhibit growth of microorganisms and higher organisms on surfaces when they are exposed to humid conditions or submerged in fresh water or marine environments. Surfaces that may be treated with the coating compositions of the present disclosure include metal, plastic, and glass surfaces of ships, structures, instrumentation, and fishing nets.

Anti-fouling coatings according to the disclosure may be water-based or oil- based solutions or emulsions. In particular embodiments, the coatings form a film that is resistant to subsequent dissolution in water, as for example in a lacquer.

However, in other embodiments, the coatings desirably degrade slowly upon exposure to water, for example, seawater, to expose a new surface and additional anti-fouling agent as the coating degrades. Such coatings may in some embodiments be planed away by relative motion of water (for example, as a ship's hull moves through water) and are known in the art as self-polishing.

Non-exhaustively, coatings incorporating the anti-fouling agents (i. e. biocides/toxicants) of the disclosure may further include binders, pigments, thickeners, extenders, hydrolysis regulators, inert diluents, plasticizers, additional anti-fouling agents, and combinations thereof.

Binders include latex, acrylics, thermoplastics, thermosets and various polymer and copolymer systems, self-polishing or otherwise. Binders also include resins, such as acrylic resins, epoxy resins, polyester resins, butyral resins, vinyl resins, polyurethane resins, urea resins, ethylene-vinylacetate resins, silicon resins, and styrene resins.

Examples of binders suitable for use in anti-fouling coatings may be found in U. S. Patent Applications No. 5,990,043 to Kugler et al. which lists polyvinyl chloride, chlorinated rubber, acrylic resins, vinyl chloride/vinyl acetate copolymer systems, butadiene/styrene rubbers, butadiene/acrylonitrile rubbers,

butadiene/styrene/acrylonitrile rubbers, drying oils, such as linseed oil, asphalt, epoxies, resin esters, modified hard resins in combination with tar or bitumen, chlorinated polypropylene, and vinyl resins.

U. S. Patent No. 5,441,743 to McGinniss, et al. describes anti-fouling compositions comprising a thermoplastic or thermosetting binder system that may be used to provide a coating which contains preferentially concentrated domains (PCD's) of the anti-fouling agents (i. e. biocides, toxicants) of the present disclosure.

Self-polishing binder systems that incorporate a combination of biologically active and biologically inactive organotin polymers are described in U. S. Patent No.

4,260,535 to Russel. Other examples of self-polishing binders include the acrylic polymer systems described in U. S. Patent No. 4,407,997 to Sghibartz that desirably may have no or low content of organotin. U. S. Patent No. 5,880,173 to Matsuda et al. describes a self-polishing binder system that incorporates a hydrolyzable metal- containing resin. U. S. Patent No. 5,472,993 to Kim et al. describes a self-polishing copolymer binder system which contains n-[(N-isothiazolonyl)-alkyl] acrylate as one of the monomers that may be utilized to desirably reduce the amount of organotin polymers in anti-fouling coatings. Hydrolyzable organosilyl acrylate copolymers are described in U. S. Patent No. 4,593,055 to Gitlitz et al.

Additional anti-fouling binders that may used for coatings that incorporate the anti-fouling agents of the present disclosure are described by Isaka et al. in U. S.

Patent No. 5,374,665, Tanaka et al. in U. S. Patent No. 5,646,198, Braeken et al. in U. S. Patent No. 4,962,135, and Ryu et al. in U. S. Patent No. 5,631,308.

Pigments that may be incorporated into coatings according to the disclosure include extender pigments and color pigments. Extender pigments include barite, barium sulfate, talc, clay, chalk, silica white, alumina white, titanium white, and bentonite. Color pigments include titanium dioxide, zirconium dioxide, zinc oxide, basic lead sulfate, tin oxide, carbon black, graphite, red iron oxide, chromium green, emerald green, phthalocyanine blue, copper rhodamine, and cuprous oxide, cuprous thiocyanate, cupric acetate, and meta-arsenate, some of which also function as anti- fouling agents.

Thickeners (viscosity regulators) include amides and amines such as nicotinamide or n-octylamine ; monobasic organic acids such as acetic, oleic or lauric acid; phosphoric acid and phosphate esters; and solid acids such as silicate or molybdate.

Inert diluents (solvents) include water ; hydrocarbons such as xylene, toluene, benzene, ethylbenzene, cyclopentane, cyclohexane, heptane, octane, mineral spirits, naptha, ligroin, and methyl isobutylketone ; ethers such as dioxane, tetrahydrofuran, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol dimethyl ether, ethylene glycol monobutyl ether, ethylene glycol dimethyl ether, ethylene glycol monomethyl ether, and diethylene glycol monoethyl ether; esters such as butyl acetate, propyl acetate, benzyl acetate, ethylene glycol monomethyl ether acetate and ethylene glycol monoethyl ether acetate; ketones such as methyl isobutylketone or ethyl isobutylketone; and alcohols such as methanol, ethanol, propanol, n-butanol, and fusel oil.

Hydrolysis regulators include chlorinated paraffin, polyvinyl ether, polypropylene, sebacate, partially hydrogenated terphenyl, polyvinyl acetate, polyalkyl (meth) acrylate, poly ether polyol, alkyd resin polyester resin, and polyvinyl chloride.

Plasticizers include tritolyl phosphate, diisooctyl phthalate, tributyl phosphate, butyl benzyl phthalate, dibutyl tartrate, and Lutenal A25.

Additional anti-fouling agents include triorganotin salts and oxides, for example, triphenyltin fluoride, tributyl tin fluoride, tributyl tin dibromosuccinate, triphenyltin chloride, tributyltin hydroxide, and tributyltin oxide. Other anti-fouling agents include di-thiocarbamate derivatives such as cuprous ethylene bis-di- thiocarbamate or 2- (N, N-dimethylthiocarbamylthio)-5-nitro thiazole, substituted isothiozolones such as halogenated N-substituted isothiazolones, teteramethylthiuram disulphide, and dichlorodiphenyltrichloroethane. Hexacyano compounds useful as anti-fouling agents are described in U. S. Patent No. 5,976,229 to Ohmura et al. A large list of insecticides that may be useful as anti-fouling agents is found in U. S. Patent No. 5,990,043 to Kugler et al.. Additional examples of anti-

fouling agents useful for the disclosed embodiments of the anti-fouling compositions may be found in the patents discussed previously in reference to binder systems.

Anti-fouling coating compositions of the present disclosure may be prepared by any method known in the art of coating formulation using machines such as ball mills, pebble mills, and roll mills to mix the ingredients.

Anti-fouling coating compositions according to the disclosure may comprise a pure anti-fouling agent (e. g., smeared as a thin layer on a window to reduce slime fouling) or a mixture wherein the anti-fouling agent is present at an effective concentration, such as from about 1% and about 80% by weight, such as between about 1% and about 50% by weight, for example, between about 5 % and about 30% by weight of the mixture. In disclosed embodiments, the anti-fouling agent is present at a concentration from about 5% to about 10% by weight. The remainder of the coating may comprise additional components selected from the group consisting of binders, pigments, thickeners, extenders, hydrolysis regulators, inert diluents, plasticizers, additional anti-fouling agents, and combinations thereof.

Persons of ordinary skill in the art will recognize that the illustrated embodiments are only particular examples and that the following claims and all embodiments that they encompass define the scope of the invention.