WO1996019464A1

WO1996019464A1 - 5-substituted-2-(thiocyanatomethylthio)-1,3,4-oxadiazoles useful as antimicrobials and antifoulants

Info

Publication number: WO1996019464A1
Application number: PCT/US1995/013493
Authority: WO
Inventors: Duane R. Romer; Ravi B. Shankar; R. Garth Pews
Original assignee: The Dow Chemical Company
Priority date: 1994-12-21
Filing date: 1995-10-10
Publication date: 1996-06-27

Abstract

Substituted 2-(thiocyanatomethylthio)-1,3,4-oxadiazoles are prepared which correspond to formula: (I) wherein R represents (a), (b), (c) or (d) wherein X represents -Br, -Cl, -F, -NO2 or -CH3 and is 0, 1 or 2. These compounds have been found to exhibit antimicrobial and marine antifouling activity in industrial and commercial applications and compositions containing these compounds are so employed.

Description

5-SUBSTITUTED-2-(THI0CYANAT0METHYLTHI0)-l,3,4-0XADIAZ0LES USEFUL AS ANTIMICROBIALS AND ANTIFOULANTS.

The present invention is directed to 5-substituted-2-(thiomethylthiocyanate)- -1 ,3,4-oxadiazole compounds, compositions containing said compounds, their preparation and their use as antimicrobial agents and marine antifoulants.

U.S. Patent 3,888,869 discloses the compound 2,5-bis(thiocyanatomethylthio)- -1 ,3,4-thiadiazole, corresponding to the formula

and the 3,5-bis(thiocyanatomethy!thio)-1 ,3,4-thiadiazole isomer thereof. The preparation of these compounds is also taught as well as their use as antimicrobial agents for the control of bacteria and fungi in various materials.

German Patent 2541 388 discloses compounds corresponding to the formula

wherein R represents C^C^ straight or branched chained alkyl and X is oxygen or sulfur The preparation of these compounds and their fungicidal use is also taught.

While the above compounds are somewhat active as antimicrobial agents, it is still desirable to identify or discover new agents. Many of the known antimicrobial as well as marine antifoulant agents have been found to be of minimal value for several reasons; these include, but are not limited to, the problem created by microbe strains developing resistance to known agents, the occurrence of undesirable interactions of certain known agents with the medium or product in which the agent is used, and the high toxicity of certain known agents to certain non-target organisms such as mammals. The present invention discloses new compounds which have high activity as antimicrobial and marine antifoulant agent.

The present invention is directed to 5-substituted-2-(thiomethylthιocyanate)- -1 ,3,4-oxadiazole compounds corresponding to the formula:

N _ N

wherein R represents

wherei n X represents -Br, -Cl, -F, -NO. or -CH.. and n is 0, 1 or 2

The present invention is also directed to antimicrobial and marine antifou ng compositions comprising an inert diluent in intimate admixture with an antimicrobially-effective amount of a compound corresponding to Formula I

The present invention is further directed to a method for inhibiting o microorganisms present in a microbial habitat which comprises contacting said microbial habitat with an antimicrobial composition comprising an inert diluent and an antimicrobially-effective amount of a compound corresponding to Formula I

The antimicrobial compositions of the present invention can also be employed to treat surfaces exposed to a marine environment in which marine organisms grow to prevent 5 the growth of said marine organisms on said surfaces

In the present specification and claims, the term "alkali metal" is employed to designate sodium, potassium, lithium or cesium

In the present specification and claims, the term "halo" is employed to designate bromo, chloro, fluoro or lodo 0 In the following process schematic formulas, certain specific alkali metals, halo groups and specific solvents are set forth These representations are only presented for convenience and are not to be considered as an indication that these specific representations are the only groups or materials which can be employed The reactions as set forth below and in the specific examples can all be carried out at room temperature in the presence of 5 conventional reaction mediums, such as for example, a 70 percent ethanol/30 percent water mixture

The 5-substιtuted-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole compounds of the present invention may be prepared by the reaction of an appropriate 5-substιtuted 2-(halomethylthιo)-1 ,3,4-oxadιazole with an alkali metal thιocyanate (AMSCN) in a reaction 0 medium The general reaction scheme for this reaction is as follows

wherein R represents furanyl, pyπdyl, or phenyl In carrying out these reactions, the oxadiazole reactaπt, such as for example, a 2-(halomethylthiol)-5-(furanyl, pyridyl, or phenyl)-1,3,4-oxadiazole and the alkali metal thiocyanate, for example, potassium thiocyanate are typically mixed together in substantially equimolar amounts in the presence of a polar, aprotic solvent, such as, for example, dimethylformamide or dimethylsulfoxide.

The reactions are typically carried out at a temperature of from 30 to 100°C. Typically, the reactants may be added to the reaction mixture in any order of addition; conventionally they are added as a solution in the solvent used for the reaction. Subsequent to the addition of the reactants, the mixing of the reaction mixture will typically be allowed to continue over a period of from 1 to 24 hours.

The reaction product resulting from the furanyl or pyridyl reactants are usually isolated by first concentrating the mixture to dryness and the taking up the residue in a solvent such as methylene chloride and washing the mixture with water and then with brine. The mixture is dried (usually over anhydrous sodium sulfate) and concentrated to give the desired product.

The reaction product resulting from the phenyl reactants may be isolated by adding a 3 to 10 volume excess of water which will precipitate the desired product. Filtration followed by washing and drying yields the desired compounds of the present invention.

The 5-phenyl compounds of the present invention may also be prepared by the reaction of an appropriately substituted 5-phenyl-1 ,3,4-oxadiazole-2-thiol corresponding to the formula:

wherein X and n are as defined hereinabove with a halomethylthiocyanate, such as chloromethylthiocyanate. The general reaction scheme for this reaction is as follows:

CN

wherein Z represents -Br, -Cl, -F or -I. The preparation of the appropriate 2-(halomethylthio)-5-(furanyl, thiofuranyl, pyridyl or phenyl)-1 ,3,4-oxadiazole starting material begins with the reaction of an appropriate 5-(furanyl, thiofuranyl, pyridyl or phenyl)-1 ,3,4-oxadiazol-2-thiol with a dihalomethane, for example, bromochloromethane in the presence of a base such as, for example, tπethylamine, pyπdine or sodium methoxide and an aprotic or protic solvent such as, for example, methylene chloride, chloroform, methanol or pyπdine. The general reaction scheme for this reaction is as follows:

wherein Z is as defined hereinabove.

In carrying out this reaction, the acid acceptor is added slowly to the 5-(2-

-thιofuranyl)-1 ,3,4-oxadιazole 2-thιol and dihalomethane in the reaction medium and the resulting solution is stirred at room temperature for a period of from 1 to 24 hours The reaction mixture is worked up by washing first with 1 M HCI, followed by a saturated aqueous sodium carbonate solution and then a brine solution The reaction mixture is dried (usually over anhydrous sodium sulfate) and concentrated to give the desired product

The 5-(furanyl, thiofuranyl or pyrιdyl)-1 ,3,4-oxadιazol-2-thιols used as a starting reactant can be conveniently prepared from the appropriate acylhydrazide

In these reactions, the appropriate acylhydrazide is stirred for 15 minutes to 1 hour at room temperature with a solution of an alkali methyl hydroxide in a medium such as methanol This mixture is then refluxed with carbon disulfide from 4 to 8 or more hours The reaction mixture is then cooled to room temperature, extracted with ether and the aqueous layer which forms is acidified to a pH of about 4 with concentrated HCI The solid which precipitates is filtered off, washed with water and dried to give the desired product The following examples illustrate the present invention and the manner by which it can be practiced but, as such, should not be construed as limitations upon the overall scope of the same

In the preparation of the compounds of the present invention, the amount of the reactants to be employed is not critical In most cases, it is preferred to employ substantially equimolar amounts of the reactants. Depending upon the specific type of reaction taking place, it may be beneficial that a given one of the reactants be present in a slight excess to obtain the highest yield of the desired product

The desired product can be separated from the reaction product of the above preparative procedures employing conventional separatory procedures known to those skilled in the art including steps of solvent extraction, filtration, water washing, column chromatography, neutralization, acidification, crystallization and distillation Since the hereinabove and hereinafter set forth compound preparation procedures employ only standard chemistry practices and it is known that slightly different reactants can require slightly different reaction parameters from those for other reactants, it is to be understood that minor modifications to the reaction parameters set forth such as the choice of the specific solvent or reaction medium employed, the use of an excess of one reactant, the use of a catalyst, the use of high temperature and/or high pressure equipment, high speed mixing and other such conventional changes are within the scope of the present invention.

The structure identity of all compounds is confirmed by proton nuclear magnetic resonance spectroscopy ('H NMR), recorded at 300 MHz; carbon nuclear magnetic resonance spectroscopy (^,3C NMR) recorded at 75 MHz; infrared spectroscopy (IR) and gas chromatography/mass spectrometry (GC/MS). All of the reactions are conducted under a positive pressure of nitrogen.

Example I: Preparation of 5-(2-Furanyl)-2-(thιomethylthiocyanate)-1 ,3,4-oxadiazole

A solution of 2-(chloromethylthiol)-5-(2-furanyl)-1 ,3-4-oxadiazole (6.32 grams (g), 0.0025 mol) and KSCN (4.9 g, 0.050 mol) in DMF (50 milliliters (mL)) was heated at 60°C. for 12 hours. The reaction mixture was concentrated to dryness and the residue was taken up in methylene chloride ( 100 mL). The mixture was washed with water followed by brine and then dried over sodium sulfate and concentrated The residue was purified by flash chromatography, eluting with 50 percent ETOAc hexanes, to give the above-named product as a yellow colored amorphous solid. ⁱ H NMR (300 MHz, CDCI₃) δ 7.67 (s, 1 H), 7.19 (bs, 1 H), 6.62 (bs, 1 H), 4.84 (s, 2H). Example IA: Preparation of 2-Chloromethylthio-5-(2-furanyl)-1 ,3,4-oxadiazole

To a solution of 5-(2-furanyl)-2-thio- 1 ,3,4-oxadiazole (5.0 g, 0.0300 mol) and bromochloromethane (5.77 g, 0.0450 mol) in 50 mL of methylene chloride was added dropwise triethylamine (2.05 g, 0.0203 mol). The solution was stirred overnight (about 16 nours) at room temperature The reaction mixture was washed first with 1 M HCI (2X 50 mL), followed by a saturated aqueous sodium bicarbonate solution and then brine The solution was dried over sodium sulfate and concentrated giving 6 32 g (84 percent) of the title compound as a viscous oil ⁱH MR (300 MHz, CDCI₃) δ 7 66 (s, 1 H), 7 17 (s, 1 H), 5 26 (s, 1 H), 5 26 (s, 2H), '3C NMR (CDCI₃) 5 160 26, 159 08, 145 81, 138 55, 1 14 43, 1 12 14 Example IB Preparation of 5-(2-Furanyl)- 1 ,3,4-oxadιazole-2-thιol

To a solution of potassium hydroxide (4 9 g, 0 087 mol) in 100 mL of methanol was added 2-furanyl-acylhydrazιde (10 0 g, 0 079 mol) The solution was stirred at 25°C for 30 minutes Carbon disulfide (12 0 g, 0 16 mol) was added and the reaction mixture was heated at reflux for 8 hours The reaction mixture was cooled to 25°C and then taken up in water (100 mL) The aqueous layer was acidified to pH 4 with concentrated HCI and the precipitate which formed was filtered off, washed with water and dried, giving 10 4 g (78 percent of theoretical) of the title compound as a white solid Example II Preparation of 5-(2-Thιofuranyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

Triethylamine (1 1 5 g, 0 1 14 mol) was added dropwise to a solution of 5-(2- -thιofuranyl)-1 ,3-4-oxadιazole-2-thιol (12 O g, 0 065 mol) and bromochloromethane (12 6 g, 0 0978 mol) in methylene chloride (150 mL) and the resulting solution was stirred overnight (about 16 hours) at room temperature The reaction mixture was washed with 1 M HCI (2X 50 mL) followed by a saturated aqueous sodium bicarbonate solution and then with brine The mixture was then dried over sodium sulfate and concentrated to give 13 3 g (88 percent yield) of 2-(chloromethylthιo)-5-thιofuranyl-1 3-4-oxadιazole as a viscous oil The oil was dissolved in DMF (50 mL) and KSCN ( 1 1 1 g, 0 1 14 mol) was added and the resulting solution was heated at 65^CC for 12 hours The reaction mixture was concentrated to dryness and the residue was taken up in methylene chloride (100 mL) The mixture was washed with water followed by brine and then dried over sodium sulfate and concentrated The residue was purified by flash chromatography, eluting with 50 percent ETOAc hexanes, to give the above-named product in a yield of 14 g ( 85 percent of theoretical) as a yellow oil ¹ H NMR (300 MHz, CDCI₃) δ 7 74 (bs, 1 H), 7 58 (bs, 1 H), 7 17 (bs, 1 H), 4 81 (s, 2H) Example IIA: Preparation of 5-(2-Thιofuranyl)-1 ,3,4-oxadιazole-2-thιol

To a solution of potassium hydroxide (10 8 , 0 193 mol) in 200 mL of methanol was added 2-thιofuranyl-acylhydrazιde (25 0 g, 0 176 mol) The solution was stirred at 25°C for 30 minutes Carbon disulfide (27 0 g, 0 352 mol) was added and the reaction mixture was heated at reflux for8 hours The reaction mixture was cooled to 25°C and then taken up in water (100 mL) The aqueous layer was acidified to pH 4 with concentrated HCI and the precipitate which formed was filtered off, washed with water and dried, giving 24 8 g (76 percent of theoretical) of the title compound as a white solid Example III Preparation of 5-(3-Pyrιdyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

A solution of 2-(chloromethylthιol)-5-(3-pyrιdyl)-1 ,3-4-oxadιazole (3 46 g, 0 0152 mol) and KSCN (2 95 g, 0 0304 mol) in DMF (25 mL) was heated at 60°C for 12 hours The reaction mixture was concentrated to dryness and the residue was taken up in methylene chloride (100 mL) The mixture was washed with water, followed by brine and then dried over sodium sulfate and concentrated The residue was purified by flash chromatography, eluting with 50 percent ETOAc hexanes, to give the above-named product in a yield of 2 85 g (75 percent of theoretical) as a yellow colored amorphous solid H NMR (300 MHz, Acetone-de) δ 9 22 (s, 1 H), 8 80 (d, J = 4 7 Hz, 1 H), 8 40 (d, J = 8 0 Hz, 1 H), 7 62 (dd, J = 8 0, 5 0 Hz, 1 H), 5 60 (s, 2H); i3C NMR (Acetone-d₆) δ 165 13, 162 45, 153 14, 147 96, 134 42, 124 60, 120 51 , 1 1 1 28, 38 15 Example MIA: Preparation of 2-Chloromethylthio-5-(3-pyridyl)- 1,3, 4-oxadiazole

To a solution of 5-(3-pyridyl)-2-thiol-1 ,3,4-oxadiazole (13.0 g, 0.0725 mol) and bromochloromethane (14.1 g, 0J09 mol) in 150 mL of methylene chlo ide was added dropwise

10 triethylamine (14.7 g, 0.145 mol). The solution was stirred overnight (about 16 hours) at room temperature. The reaction mixture was washed first with 1M HCI (2X 50 mL), followed by a saturated aqueous sodium bicarbonate solution and then brine. The solution was dried over sodium sulfate and concentrated giving 3.46 g (21 percent) of the title compound as a yellow oil. ¹H NMR (300 MHz, CDCI₃) δ 9.24 (s, 1 H), 8.79 (d, J = 4.9 Hz, 1 H), 8.33 (d, J = 8.0 Hz, 1 H), 7.50

15 (dd, J = 7.7, 4.7 Hz, 1H), 5.33 (s, 2H); ^,3C NMR (CDCI₃) δ 164.1 1 , 161.59, 152.31 , 147.37, 133.68, 123.57, 1 19.53, 45.12. Example 1MB: Preparation of 5-(3-Pyridyl)- 1 ,3, 4-oxadiazole-2-thiol

To a solution of potassium hydroxide (1 1.2 g, 0.200 mol) in 200 m of methanol ^ was added 3-pyridine-acylhydrazide (25.0 g, 0.182 mol). The solution was stirred at 25°C. for 30 minutes. Carbon disulfide (28.1 g, 0.365 mol) was added and the reaction mixture was heated at reflux for 8 hours. The reaction mixture was cooled to 25°C. and then taken up in water (100 mL). The aqueous layer was acidified to pH 4 with concentrated HCI and the precipitate which formed was filtered off, washed with water and dried, giving 24.2 g (74 percent of theoretical) 30 of the title compound as a white solid.

Example IV: Preparation of 5-Phenyl-2-(thiomethylthiocyanate)- 1 ,3,4-oxadiazole

35 SCH2SCN

A solution of 2-(chloromethylthιo)-5-phenyl-1 ,3-4-oxadiazole (3.0 g, 0 013 mol) and KSCN (3.9 g, 0.040 mol) in D F (30 mL) was heated at 60°C. for 16 hours The reaction mixture was allowed to cool to room temperature and water (100 mL) was slowly added dropwise with stirring. The resulting slurry was stirred for 30 minutes, and the precipitate was filtered off. The solid was washed with water, dried and crystallized from ethanol to give 2.51 g (76 percent yield) of the above-named product as a white powder which melted at 95- -96^βC. TLC RF 0.18 (20 percent ETAc hexanes); i H NMR (300 MHz, CDCI₃) δ 8.02 (d, J = 6 4 Hz, 2H), 7.52 (d, J = 7.4 Hz, 2H), 4.80 (s, 2H); 13C NMR (75 MHz, CDCI3) δ 166 69, 160.79, 132.00, 129.00, 126.66, 122.87, 1 10.64, 37.33. Example V: Preparation of 5-(4-Methylphenyl)-2-(thiomethylthiocyanate)-1 , 3, 4-oxadιazole

The preparative procedure of Example I was used to react 2-(chloromethylthio)-5-(4-methylphenyl)-1 ,3,4-oxidiazole and potassium thiocyanate in dimethylformamide. The product was crystallized from ethanol and recovered in a yield of 90 percent of theoretical as a white solid which melted at 1 16-1 17°C. ' H NMR (300 MHz, CDCI₃) δ 7.90 (d, J = 8.0 Hz, 2H), 7 31 (d, J = 8.0 Hz, 2H), 4.80 (s, 2H), 2 43 (s, 3H); ¹3C NMR (75 MHz, CDCI3) δ 166.88, 160.37, 142.66, 129 71 , 126.64, 120.1 1, 1 10.69, 37.37, 21.74; MS (El) m/e 265 (M + 2, 6), 263 (M + , 62), 205 (M-SCN, 1 1), 159 (M-SCH.SCN, 100) Example VI: Preparation of 5-(4-Chlorophenyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

Following the preparative procedure of Example I, 2-(chloromethylthio)-5-(4-chlorophenyl)- 1 ,3,4-oxadiazole and potassium thiocyanate were reacted in dimethylformamide The product was crystallized from ethanol and recovered in a yield of 91 percent of theoretical as a white solid which melted at 120- 122°C ¹ H NMR (300 MHz, CDCI3) δ 7 96 (d, J = 8 3 Hz, 2H), 7.50 (d, J = 8.5Hz, 2H), 4.80 (s, 2H), 3C NMR (75 MHz, CDCI₃) δ 165 90, 161.08, 138.34, 129 43, 127 94, 121.33, 1 10 56, 37 32; MS (El) m/e 285 (M + 2, 23), 283 (M + , 56), 225 (MSCN, 20), 179 (M-SCH.SCN, 100) Example VII: Preparation of 5-(2-Chlorophenyl)-2-(thιomethylthιocyanate)- -1 ,3,4-oxadιazole

The preparative procedure of Example I is used to react 2-(chloromethylthιo)-5-(2-chlorophenyl)-1 ,3,4-oxadιazole and potassium thiocyanate in dimethylformamide The product was crystallized from ethanol and recovered in a yield of 83 percent of theoretical as a pale yellow solid which melted at 84-86°C TLC RF 0 22 (20 percent ETOAc/hexanes), ^" H NMR (300 MHz, DMSO-d₆) δ 8 03 (d, J = 7 1 Hz, 1 H), 7 74-7.56 (m, 3H), 5 17 (S, 2H); ⁱ 3C NMR (75 MHz, DMSO-d₆) δ 163 76, 161 50, 133 21 , 131 60, 131 10, 130 95, 127 63, 121 74, 1 1 1 63, 37 52; MS (El) m/e 285 (M + 2, 15), 283 (M + , 34), 179 (M-SCH^SCN, 100) Example VIII preparation of 5-(4-Nιtrophenyl)-2-(thιomethylthιocyanate)- -1 ,3,4-oxadιazole

The preparative procedure of Example I is used to react 2-(chloromethylthιol)-5-(4-nιtrophenyl)-1 ,3,4-oxadιazole and potassium thiocyanate in dimethylformamide The product was crystallized from ethanol and recovered in a yield of 91 percent of theoretical as a white solid which melted at 85-88°C ' H NMR (300 MHz, DMSO-d₆) δ 8 43 (d, J = 8 6 Hz, 2H), 8 3 (d, J = 8 5 Hz, 2H), 5 19 (s, 2H); 13 NMR (75 MHz, DMSO-d₆) δ 164 35, 162 23, 149 06, 128 14, 127.80, 124 40, 1 1 1 69, 37 60; MS (El) m/e 294 (M + , 44), 190 (M-SCH₂SCN, 99), 236 (M-SCN, 54) Example IX Preparation of 2-(Chloromethylthιo)-5-phenyl-1 ,3,4-oxadιazole

A mixture was prepared by adding triethylamine (2 6 g, 0 026 mol) in CH2CI2 (5 mL) dropwise to a slurry of 5-phenyl- 1 ,3, 4-oxadιazol-2-thιol (3 O g, 0 017 mol) and bromochloromethane (2.8 g, 0.021 mol) in CH₂Cl2 (30 mL) at 0°C. The resulting mixture is stirred overnight at room temperature. The reaction mixture is washed with 20 mL of 1 M HCI, then with 20 mL of aHCθ3 and 20 mL of brine. The washed material is then dried over Na2S0 and concentrated to give the above-named product as a light yellow oil in a yield of 3.0 g (92 percent of theoretical). TLC RF 0.36 (20 percent ETAc/hexanes); ■ H NMR (300 MHz, CDCI₃) δ 7.97-7.95 (m, 2H), 7.47-7.45 (m, 3H), 5.31 (s, 2H); 13C NMR (75 MHz, CDCI3) δ 165.85, 160.40, 131.42, 128.53, 126.64, 45.26. Example X: Preparation of 2-(Chloromethylthio)-5-(4-methylphenyl)-1 , 3, 4-oxadiazole

The preparative procedure of Example VI is used to react 5-(4- -methylphenyl)-1 ,3,4-oxadiazol-2-thιol and bromochloromethane The recovered product is obtained in a yield of 96 percent of theoretical as a light yellow solid TLC RF 0.50 (30 percent ETAc hexanes); ¹ H NMR (300 MHz, CDCI3) δ 7 84 (d, J = 8.2 Hz, 2H), 7 25 (d, J = 8.2 Hz, 2H), 5 29 (s, 2H); 2.38 (s, 3H); 13c NMR (75 MHz, CDCI₃) δ 166.10, 160.07, 142.06. 129 27, 126.17, 1 19 91 , 45.28, 21.34. Example XI: Preparation of 2-(Chloromethylthio)-5-(4-chlorophenyl)-1 , 3, 4-oxadιazole

The preparative procedure of Example VI is used to react 5-(4- -chlorophenyl)-1 ,3,4-oxadiazol-2-thiol and bromochloromethane The recovered product is obtained in a yield of 86 percent of theoretical as a white solid TLC RF 0 72 (30 percent ETAc hexanes); ¹ H NMR (300 MHz, CDCI.) δ 7.96 (d, J = 8.5 Hz, 2H), 7 48 (d, J = 8.5 Hz, 2H), 5 28 (S, 2H); 13C NMR (75 MHz, CDCI ₃) δ 165.51 , 161.03, 138.07, 129 32, 127 86, 121 53, 45.23 Example XII: Preparation of 2-(Chloromethylthio)-5-(2-chlorophenyl)-1 ,3,4-oxadiazole

The preparative procedure of Example VI is used to react 5-(2- -chlorophenyl)-1 ,3,4-oxadiazol-2-thiol and bromochloromethane. The recovered product is obtained in a yield of 53 percent of theoretical as a yellow oil, which slowly solidified upon standing. TLC RF 0.42 (20 percent ETAc/hexanes); ¹ H NMR (300 MHz, CDCI,) δ 7.94 (d, J = 7.5 Hz, 1 H), 7.54-7.37 (m, 3H), 5.34 (s, 2H); ^{, J}C NMR (75 MHz, CDCI,) δ 164.18, 161.18, 132.37, 132.22, 130.75, 130.43, 126.70, 127.86, 121.85, 45.16. Example XIII: Preparation of 2-(Chloromethylthio)-5-(4-nitrophenyl)-1 ,3,4-oxadiazole

The preparative procedure of Example VI is used to react 5-(4- -nitrophenyl)-1 ,3,4-oxadiazol-2-thiol and bromochloromethane. The recovered product is obtained in a yield of 77 percent of theoretical as a yellow solid. 'H NMR (300 MHz, CDCI.) δ 8.39 (d, J = 8.6 Hz, 2H), 8.24 (d, J = 8.95 Hz, 2H), 5.34 (s, 2H); ¹ C NMR (75 MHz, CDCI,) δ 164.48, 162.39, 149.30, 128.42, 127.49, 124.21 , 45.06.

The compounds of this invention are useful as antimicrobial additives to such industrial products as styrene-butadiene latexes used for paper coatings, paints, inks, adhesives, soaps, cutting oils, textiles, and paper and pigment slurries. The compounds are also useful as antimicrobial additives in such personal care products as hand creams, lotions, shampoos, and hand soaps. A further advantage of this invention is its cost-effectiveness for applications which need to have an antimicrobial continuously replenished, such as in cooling towers and pulp and paper mills.

As appreciated in the art, not all of the compounds disclosed herein are active at the same concentrations or against the same microbial species. That is, there is some compound-to-compound variation in antimicrobial potency and spectrum of antimicrobial activity.

The present invention is also directed to a method for inhibiting microorganisms which comprises contacting said microorganisms or habitat thereof with an effecti e amount of the compounds of this invention.

The antimicrobial compounds of this invention may be added directly to aqueous formulations susceptible to microbial growth, either undiluted or dissolved, in inert diluents such as organic solvents such as glycols, alcohols, or acetone. They may also be added alone or in combination with other preservatives. As used herein, the term "microorganism" is meant to refer to bacteria, fungi, viruses, algae, subviral agents and protozoa.

As used herein, the term "antimicrobially-effective amount" refers to that amount of one or a mixture of two or more of the compounds, or of a composition comprising such compound or compounds, of this invention needed to exhibit inhibition of selected microorganisms. Typically, this amount varies from providing 1 part per million (ppm) to 5,000 ppm by weight of the compound to a microbial habitat being contacted with the compound. Such amounts vary depending upon the particular compound tested and microorganism treated. Also, the exact concentration of the compounds to be added in the treatment of industrial and consumer formulations may vary within a product type depending upon the components of the formulation. A preferred effective amount of the compound is from 1 ppm to 500 ppm, more preferably from 1 ppm to 50 ppm by weight, of a microbial habitat.

The term "microbial habitat" refers to a place or type of site where a microorganism naturally or normally lives or grows. Typically, such a microbial habitat will be an area that comprises a moisture, nutrient, and/or an oxygen source such as, for example, a cooling water tower or an air washing system.

The terms "inhibition", "inhibit" or "inhibiting" refer to the suppression, stasis, kill, or any other interference with the normal life processes of microorganisms that is adverse to such microorganisms, so as to destroy or irreversibly inactivate existing microorganisms and/or prevent or control their future growth and reproduction

The antimicrobial activity of the compounds of the present invention is demonstrated by the following techniques.

The minimum inhibitory concentration (MIC) for the compounds of the present invention is determined for 9 bacteria, using nutrient agar; and 7 yeast and fungi, using malt yeast agar. A one percent solution of the test compound is prepared in a mixture of acetone and water. Nutrient agar is prepared at pH 6.8, representing a neutral medium, and at pH 8 2, representing an alkaline medium. The nutrient agars are prepared by adding 23 g of nutrient agar to one-liter of deionized water In addition, the alkaline medium is prepared by adjusting a 0.04 M solution of N-[tris-(hydroxymethyl)methyl]-glycine buffered deionized water with concentrated sodium hydroxide to a pH of 8.5. Malt yeast agar is prepared by adding 3 g yeast extract and 45 g malt agar per liter of deionized water. The specific agar is dispensed in 30 mL aliquots into 25 x 200 mm test tubes, capped and autoclaved for 15 minutes at 1 15°C. The test

5 tubes containing the agar are cooled in a water bath until the temperature of the agar is 48°C Then, an appropriate amount of the one percent solution of the test compound is added (except in the controls where no compound is added) to the respective test tubes so that the final concentrations are 500, 250, 100, 50, 25, 10, 5, 2.5, 1.0 and zero parts per million of the test compound in the agar, thus having a known concentration of test compound dispersed

1 o therein. The contents of the test tubes are then transferred to respecti e petn plates After drying for 24 hours, the petri plates containing nutrient agar are inoculated with bacteria and those containing malt yeast agar are inoculated with yeast and fungi.

The inoculation with bacteria is accomplished by using the following procedure. Twenty-four hour-cultures of each of the bacteria are prepared by incubating the respective

15 bacteria in tubes containing nutrient broth for 24 hours at 30°C in a shaker. Dilutions of each of the 24 hour-cultures are made so that nine separate suspensions (one for each of the nine test bacteria) are made, each containing 10⁸ colony forming units (CFU) per mL of suspension of a particular bacteria. Aliquots of 0.3 mL of each of the bacterial suspensions are used to fill the individual wells of Steer's Replicator. For each microbial suspension, 0.3 mL was used to fill

20 three wells ( that is, three wells of 0.3 mL each) so that for the nine different bacteria, 27 wells are filled. The Steer's Replicator is then used to inoculate both the neutral and alkaline pH nutrient agar petri plates.

The inoculated petri plates are incubated at 30°C for 48 hours and then read to determine if the test compound which is incorporated into the agar prevented growth of the

25 respective bacteria

The inoculation with the yeast and fungi is accomplished as follows Cultures of yeast and fungi are incubated for seven days on malt yeast agar at 30°C. These cultures are used to prepare suspensions by the following procedure. A suspension of each organism is prepared by adding 10 mL of sterile saline and 10 microliters of octylphenoxy polyethoxy

30 ethanol to the agar slant of yeast or fungi The sterile saline/octylphenoxy polyethoxy ethanol solution is then agitated with a sterile swab to suspend the microorganism grown on the slant Each resulting suspension is diluted into sterile saline (1 part suspension: 9 parts sterile saline) Aliquots of these dilutions are placed in individual wells of Steer's Replicator and petri plates inoculated as previously described. The petri plates are incubated at 30°C and read after

35 48 hours for yeast and 72 hours for fungi.

Table I lists the bacteria, yeast and fungi used in the MIC test described above along with their respective American Type Culture Collection (ATCC) identification numbers. TABLE I

Organisms used in the Minimum

5

In Tables II and III, the MIC values of the compounds of the present invention as compared to the MIC of a standard commercial preservative (with 1-(3-chloroallyl)-3,5,7-tπaza- -1-azonιaadamantane chloride as the active agent, and referred to in Tables II and III as "STANDARD") are set forth for the bacteria organisms and yeast/fungi organisms which are listed in Table I. 0

5

TABLE III

Minimum Inhibitory Concentrations for Test Compounds in Yeast/Fungi Species (in ppm) COMPOUND ORGANISMS EXAMPLE NO. An Ca Pc Sc Tv Ap Fo

STANDARD >500 >500 >500 500 >500 >500 >500

I <10 <10 <10 <10 25 <10 <10 ^π <1 5 <1 <1 25 <1 2.5

III 2.5 25 2.5 25 50 <1 5

IV <1 5 <1 <1 25 <1 2.5

V <1 2.5 <1 <1 10 < 1 5

VI <1 2.5 <1 <1 10 <1 5 VII 2.5 2.5 <1 < 1 10 < 1 5

VIII 2.5 5 <1 2.5 10 <1 5

The present invention is also directed to a method for inhibiting marine organisms. The term "marine organisms" is meant to include marine animals, such as barnacles, serpulid, bryozoa, oysters and hydroids, and marine plahts, such as green algae and brown algae. The method for inhibiting marine organisms comprises contacting a surface exposed to a marine environment in which marine organisms grow with a marine antifouhng effective amount of the compound of this invention.

As appreciated by those skilled in the art, not all of the compounds disclosed herein are active at the same concentrations or against the same marine organism species That is, there may be some compound-to-compound variation in marine antifouhng potency and spectrum of marine antifouling activity. Furthermore, the level of a specific compound's marine antifouling activity may be dependent on various factors including the specific materials with which the compound is formulated.

As used herein, the term "marine antifouling effective amount" refers to that amount of one or a mixture of two or more of the compounds of this invention needed to exhibit inhibition of selected marine organisms Typically, this amount vanes from providing 1 weight percent to 30 weight percent of the compound to a marine antifouling composition which is used to treat a surface exposed to a marine environment in which marine organisms live or grow. Such amounts vary depending upon the particular compound tested and marine organism to be treated Also, the exact concentration of the compounds to be added in the preparation of industrial and consumer formulations may vary within a product type depending upon the components of the formulation A composition comprising a marine antifouling effective amount of the compound will also comprise an inert diluent which may be, for example, in the form of a paint. Particularly preferred are those paints having a vinyl resin binder such as, for example, a plasticized polyvinyl chloride or a polyvinyl chloride-polyvinyl acetate type. Preferably, the

5 binders are formulated as latexes or emulsions. In a paint composition, the compound of the present invention is preferably used in an amount from 1 to 30 weight percent and, most preferably, from 10 to 25 weight percent. In addition to vinyl resin binder paints, epoxy and polyurethane binder paints containing the compound may also be useful. Coatings and films prepared from paints comprising the compound of the present invention typically remain

10 substantially free from build-up of marine organisms for periods ranging from 3 to 12 months, depending upon the concentration of the compound and the thickness of the applied coating or film.

The term "a surface exposed to a marine environment" refers to a surface where a marine organism naturally or normally lives or grows Typically, such a surface will be an area

_] 5 that is in continual or periodic contact with a marine environment such as an ocean or other body of water. Typical surfaces include, for example, a ship hull.

The marine antifouling activity of the compounds of the present invention is demonstrated by the following techniques.

Test panels are prepared from clear, rigid polyvinyl chloride film that is

20 0.381 x 10 ³ m thick and has one textured surface The test panels are 0.1524 m by 0.1524 m squares that have 0 00635 m holes punched at corners on 0.127 m centers. A 0.102 square template, with a 0.067 m diameter hole at the center, is attached to the center of the textured surface of the test panels.

A candidate marine antifoulant compound (1 0 g) is stirred into a resinous latex

25 binder (9 O g) A portion of the compound/binder mixture (1 5 g) is added to the center of the test panel and uniformly spread over the circular area inside the template

Water is added dropwise as needed to properly spread the compound/binder mixture. The template prevents the compound/binder mixture from spreading beyond the uncovered area. The test panel is allowed to sit for between 10 to 30 minutes until the edge of

30 the spread compound/binder mixture has dried The template is then removed The test panel is then allowed to dry for 8 to 12 hours at room temperature

Two test panels are prepared for each candidate marine antifoulant compound Two control test panels are also prepared by only treating with the resinous latex binder. One test panel of each candidate marine surfactant compound is attached over a white background

35 to the topside of an exposure support apparatus The second test panel is attached over a black background to the underside of the exposure support apparatus The exposure support apparatus is placed horizontally 0 0254 m under a marine surface with the white background topside facing up The exposure support apparatus is exposed to the marine environment for both 6 and 10 weeks during which time the control test panels become substantially covered with mature marine organism growth on both the topside and underside exposures.

After being removed from the exposure support apparatus, each test panel is inspected and rated for marine organism growth on both the treated and untreated areas of

5 the test panel. The marine organisms present on the treated and untreated areas are noted. The presence of algae spores and bacterial slime are noted but not included in rating each test panel. The test panels are rated on a scale from 10 (representing completely free of marine organism growth) to 0 (representing completely covered with marine organism growth). In Table IV, the marine antifouling rating values for some of the active

10 compounds of the present invention are set forth, as well as the ratings for control panels (with no marine antifouling compound and referred to as "Control ").

In addition, test panels were prepared using tributyl tin oxide, a known marine antifouling compound. One set of such panels used the tributyl tin oxide in a commercially available ship-hull paint (referred to in Table IV as "STANDARD II ") which was employed in the

15 same manner as the resinous latex binder used on the other test panels. A second set of such panels used the tributyl tin oxide at a 10 percent concentration in the resinous latex binder (referred to in Table IV as "STANDARD III").

TABLE IV

20 Marine Antifouling Rating for Test Compounds

35

Claims

CLAIMS:

1 A compound corresponding to the formula

wherein R represents

wherein X represents -Br, -Cl, -F, -NO or -CH and n is 0, 1 or 2

2 The compound of Claim 1 which is 5-(2-furanyl)-2-(thιomethyl- thιocyanate)-1 ,3,4-oxadιazole

3 The compound of Claim 1 which is 5-(2-thιofuranyl)-2- (thiomethyl- thiocyanate)- 1 ,3,4-oxadιazole

4 The compound of Claim 1 which is 5-(3-pyπdyl)-2( thiomethyl- thιocyanate)-1 ,3,4-oxadιazole

5 The compound of Claim 1 which is 5-phenyl-2- ( thiomethyl- thιocyanate)-1 ,3,4-oxadιazole

6 The compound of Claim 1 wherein n is 0

7 The compound of Claim 6 wherein X is -CI

8 The compound of Claim 7 which is 5-(2-chlorophenyl)-2-(thιo- methylthιocyanate)-1 ,3,4-oxadιazole

9 The compound of Claim 7 which is 5-(4-chlorophenyl)-2-(thιo- methylthιocyanate)-1 ,3,4-oxadιazole

10 The compound of Claim 6 wherein X is -N0₂

1 1 The compound of Claim 10 which is 5-(4-nιtrophenyl)-2(thιo- methylthιocyanate)-1 ,3,4-oxadιazole

12 The compound of Claim 6 wherein X ιs -CH₃

13 The compound of Claim 12 which is 5-(4-methylphenyl)-2- -(thιomethylthιocyanate)-1 ,3,4-oxadιazole

14 An antimicrobial composition comprising an inert diluent and an antimicrobially-effective amount of a compound corresponding to the formula

wherein R represents

wherein X represents -Br, -Cl, -F, -N0₂ or -CH₃ and n is O, 1 or 2.

15. The composition of Claim in which the compound is 5-(2- -furanyl)-2-(thiomethylthιocyanate)-1 ,3,4-oxadιazole.

16. The composition of Claim 14 in which the compound is 5-(2- -thiofuranyl)-2-(thiomethylthiocyanate)-1 ,3,4-oxadiazole.

17. The composition of Claim 14 in which the compound is 5-(3-

-pyridyl)-2-(thιomethylthiocyanate)-1 ,3,4-oxadiazole.

18. The composition of Claim 14 in which the compound is 5-phenyl-2-(thiomethylthιocyanate)-1 ,3,4-oxadιazole.

19. The composition of Claim 14 wherein n is 0.

20. The composition of Claim 19 wherein X is -Cl

21. The composition of Claim 20 in which the compound is 5-(2- -chlorophenyl)-2-(thιomethylthiocyanate)-1 ,3,4-oxadiazole.

22. The composition of Claim 20 in which the compound is 5-(4- -chlorophenyl)-2-(thiomethylthiocyanate)-1 ,3,4-oxadiazole.

23. The composition of Claim 14 wherein X is -N0₂-

24. The composition of Claim 23 in which the compound is 5-(4- -nitrophenyl)-2-(thιomethylthiocyanate)-1 ,3,4-oxadiazole.

25. The composition of Claim 14 wherein X is -CH₃.

26. The composition of Claim 25 in which the compound is 5-(4- -methylphenyl)-2-(thιomethylthιocyanate)- l ,3,4-oχadιazole

27. The composition of Claim 14 wherein the compound is present in the composition in an amount sufficient to provide from 1 part per million to 5,000 parts per million by weight of the compound to an antimicrobial habitat that is contacted with the composition. 28. A method for inhibiting microorganisms in a microbial habitat comprising contacting said microbial habitat with an antimicrobially-effective amount of a compound corresponding to the formula:

N _ N

// ^

SCH2SCN

wherein R represents

wherein X represents -Br, -Cl, -F, -N0₂ or -CH₃ and n is 0, 1 or 2 29 The method of Claim 28 in which the compound is 5-(2-

-furanyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

30 The method of Claim 28 in which the compound is 5-(2- -thιofuranyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

31 The method of Claim 28 in which the compound is 5-(3- -pyrιdyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

32 The method of Claim 28 in which the compound is 5-phenyl-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

33 The method of Claim 28 wherein n is 0

34 The method of Claim 33 wherein X is -Cl 35 The method of Claim 34 in which the compound is 5-(2-chloro- phenyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

36 The method of Claim 34 in which the compound is 5-(4-chloro- phenyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

37 The method of Claim 33 wherein X is -N0₂ 38 The method of Claim 37 in which the compound is 5-(4-nιtro- phenyl)-2-(thιomethylthιocyanate)-1 ,3,4-oxadιazole

39 The method of Claim 33 wherein X is -CH₃

40 The method of Claim 25 in which the compound is 5-(4-methyl- phenyl)-2-(thιomethylthιocyanate)- 1 ,3,4-oxadιazole 41 The method of Claim 28 wherein the compound is used in an amount to provide from 1 part per million to 5,000 parts per million by weight of the compound to a microbial habitat being contacted with the composition

42 A method for preventing the growth of marine organisms on a surface exposed to a marine environment in which marine organisms grow comprising contacting said surface with a marine antifouling effective amount of a compound corresponding to the formula

N _ N

wherein R represents

wherein X represents -Br, -Cl, -F, -N0₂ or -CH₃ and n is O, 1 or 2.

43. The method of Claim 42 in which the compound is 5-phenyl-2-(thio- methylthiocyanate)-1 ,3,4-oxadiazole.

44. The method of Claim 42 wherein n is O.

45. The method of Claim 44 wherein X is -Cl. o

46. The method of Claim 45 in which the compound is 5-(2-chlorophenyl)-2-(thio- methylthiocyanate)-1 ,3,4-oxadiazole.

47. The method of Claim 45 in which the compound is 5-(4-chlorophenyl)-2-(thιo- methylthiocyanate)-1 ,3,4-oxadiazole.

48. The method of Claim 44 wherein X is -N0₂. 5

49. The method of Claim 44 wherein X is -CH₃.

50. The method of Claim 49 in which the compound is 5-(4-methylphenyl)-2-(thio- methylthiocyanate)-1 ,3,4-oxadiazole.

51. The method of Claim 42 wherein the compound is contacted with the surface in an amount from 1 to 30 weight percent of a composition comprising an inert diluent in 0 admixture with the compound.

5

0

5