SUBSTITUTED AROMATIC BIOLOGICALLY ACTIVE COMPOUND, A PROCESS FOR ITS PREPARATION AND ITS USE AS BIOCIDE

Abstract: A method for protecting a medium from attack by micro-organisms by addition of a biocide containing an aromatic ring carrying a thiocyanate and a carbamoyl group in ortho-relationship to each other. Also claimed is the biocide in which either the ring system or the carbamoyl group carries another substituent. The biocide is parti­ cularly useful in aqueous media such as cooling water, paper mill liquor, aqueous oil emulsions and water-based paints or paint films derived therefrom.

Description: This invention relates to a method for protecting a medium from attack by bacteria and fungi and to materials having antibacterial and antifungal activity. According to the present invention there is provided a method for protecting a medium from attack by microorganisms which comprises incorporating intc the medium a biologically-active compound containing an aromatic ring on which are substituted, in ortho-relationship to one another, a thiocyanate group and a carbamoyl group. Aqueous media containing organic species dissolved or dispersed therein are particularly prone to attack by microbes, such as bacteria1 fungi and algae, and aqueous-based paints, oil emulsions, adhesives and like systems are particularly suitable for protection by the present invention. Closed aqueous systems, such as cooling towers and the water systems of paper mills, are also susceptible to microbial attack and may be protected by these biologically-active compounds. Solid media such as paint films or wood may also be protected according to the present invention by incorporating the compound in a paint or impregnation of the medium by the compound. The aromatic ring, which may be part of a fused ring system, may be 5 or-6 membered and may contain a hetero-atom, such as oxygen but preferably sulphur or nitrogen. Examples of suitable aromatic rings and ring systems are benzene, pyridine, thiophene, pyrrole, naphthalene, quinoline, pyrimidine, thianaphthene and benzthiazole. A preferred biologically-active compound has the general formula: EMI2.1 Formula I wherein X is C, S, N or 0 Z is C, N or a direct link R is H, C1 to C20alkyl, cycloalkyl, aryl, aralkyl or C3 to C20alkenyl and R1 and R2 are each independently H, halo, nitro, cyano, thiocyanato, C1 to C16alkyl or C1 to Ckalkoxy or R1 and R2 together with the ring to which they are attached form a fused ring system. It is preferred that at least one of X and Z is a carbon atom and more preferably that both are carbon atoms. Where R is or contains an alkyl group this is preferably a lower alkyl group (C1-C4) and where R is an alkenyl group this is preferably a lower alkenyl group (C3-C6). Where R is an aryl or an aralkyl group the aryl radical is preferably phenyl. Where R1, R2 and the benzene ring together form a fused ring system this is preferably a naphthalene, quinoline, benzthiazole, benzisothiazole or benzthiophene system. It is also preferred that only one of R1 and is thiocyanato. The alkyl, alkenyl, aryl and aralkyl groups represented by B, R1 and R2 may be substituted. Preferred substituents for the substituted alkyl and alkenyl groups are hydroxy, alkoxy and halo and preferred substituents for the substituted aryl and aralkyl groups are alkyl, alkoxy, nitro and halo. Examples of the preferred lower alkyl and substituted lower alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-hydroxyethyl, 2-chloroethyl and 2-methoxyethyl. Examples of the preferred substituted aryl groups are 2-, 3- and 4-tolyl, 2-, 3- and 4-anisyl, 2-, 3- and 4-nitrophenyl and 2-, D- and 4-chloroohenyl. Examples of the preferred substituted aralkyl groups are benzyl and 2-phenylethyl. Examples of the preferred lower alkenyl radicals are propenyl and butenyl. At least one of the alkyl groups represented by R1 and R2 may contain up to 16 carbon atoms in order to provide some lipophilicity. The alkoxy group represented by R1 or 32 is preferably lower (C1 to C4) alkoxy. 1+ The quantity of the biologically-active compound which is used will depend upon the nature of the medium it is desired to protect and the organism or organisms to which the medium is susceptible. In general, the quantity is from 5 to 50,000 parts per million (ppm) by weight, based on the weight of the medium being protected and is more preferably from 50 to 10,000 ppm, by weight. Where the medium is a liquid, the compound may be added in the solid state but it is preferably incorporated in the form of a solution in a suitable solvent, e.g. a lower alkanol or a solvent/water mixture or as a suspension or dispersion in water. Where an aqueous dispersion is used; this preferably includes a dispersing agent, e.g. the sodium salt of a naphthalene sulphonic acid/formaldehyde condensate, to assist in dispersing the compound evenly throughout the medium. Solid media may be protected either by impregnation or a superficial coating with the compound. Paint films may be protected by incorporating the compound in the paint before it is applied to a surface. The method according to the invention is particularly useful where the medium to be treated is a paint film or a water based paint, e.g. an emulsion paint. Infection-susceptible media, especially paint films and water-based paints protected against infection by micro-organisms by the incorporation therein of a biologically-active compound, as herein before defined, constitute a further feature of the present invention. According to a further feature of the invention we provide the novel biologically-active compound-as herein before defined which contains an aromatic ring on which are substituted,in ortho-relationship to one another, a thiocyanate group and a carbamoyl group provided that, where the compound comprises a single benzene ring, either the benzene ring or the carbamoyl group carries at least one substituent other than hydrogen. The aromatic ring, which may be part of a fused ring system, may be 5- or 6-membered and may contain a hetero-atom, preferably sulphur or nitrogen. Examples of suitable aromatic rings and ring systems are benzene, pyridine, thiophene, pyrrole, naphthalene, quinoline, pyrimidine, thianaphthene and benzthiazole. A preferred novel biologically-active compound according to this feature of the invention has the general formula I wherein R, 31 and R2 have the above defined meanings provided that at least one of R, R1 and R2 is a group other than hydrogen. The biologically-active compound may be prepared from a compound of the formula: EMI4.1 Formula II by conversion of the amino group into a thiocyanato group and the carboxylic acid group into a carbamoyl group. These operations may be carried out in either order. The intermediate compound of the formula: EMI5.1 Formula III is novel and forms a further feature of the present invention. It may be prepared by diazotisation of the amino group in the compound of Formula II followed by treatment of the diazonium compound with an alkali metal or ammonium thiocyanate. The compound of Formula III may be converted into the compound of Formula I by treatment with a chlorinating agent such as thionyl chloride or phosphorus pentachloride to convert the COOH group into a COC1 group followed by reaction with an amine of the formula RNH2 or ammonia, wherein R has the meaning hereinbefore defined. A preferred method of preparing the biologicallyactive compounds however is by reaction of a compound containing an aromatic ring fused to a 1,2-isothiazolin-3-one ring or a substituted derivative thereof, e.g. 1,2-benzisothiazolin-3-one with an alkali metal or ammonium bisulphite to form a compound in which the aromatic ring carries a carbamoyl group and a thiosulphonate group in ortho relationship to each other and converting the thiosulphonate group into a thiocyanate group by reaction with an alkali metal or ammonium cyanide. The intermediate thiosulphonate compounds in which either the carbamoyl group or the aromatic ring carries at least one substituent other than hydrogen are a further feature of the present invention and some of them have significant biological activity particularly against fungi. A preferred thiosulphonate compound has the formula: EMI6.1 Formula IV wherein X, Z, R, R1 and R2 have the meanings hereinbefore defined and M is an alkali metal or an ammonium ion. The preferred compound is prepared by reaction of a compound according to the formula: EMI6.2 Formula V wherein X, Z, R, 31 and R2 have the above defined meanings with an alkali metal or ammonium bisulphite in an aqueous or alcoholic medium at a temperature from 0 to 10000. The preferred thiosulphonate compound may be converted into the equivalent thiocyanate compound by-reaction with an alkali metal or ammonium cyanide at a temperature from 0 to 100. ExampLe 1 Preparation of 2-thiocyanato benzamide( 2Th) (a) Preparation of Sodium 2-carbamoyl-benzene-thiosulphonate 75 parts sodium bisulphite liquor (40%), 16 parts benzisothiazolone and 25 parts water, are stirred at 45-50 C for three (3) bours. The solution is filtered hot to remove impur ties, cooled to room temperature; the precipitated product is filtered off and dried in the vacuum oven. The crude product is recrystallised from ethanol, filtering hot to remove insoluble sodium sulphate yielding 7 parts of a white crystalline solid product. Elemental analysis of the product gave the following results (theoretical values in brackets): C 32.5 (32.9); H 2.4 (2.5); N 5.5 (55); S 24.8 (25.1); Na 8.8 (9.0); IR analysis showed stretches at 3200 cm , 3380 cm , 1675 cm , 1415 cm -1 730 x 750 cm indicative of a CONH2 group and an ortho disubstituted benzene ring. (b) Conversion of thiosulphonate groun to a thiocyanate group 6.83 parts of the product of (a), 1.7 parts of potassium cyanide are both dissolved in the minimum amount of water. Solution of potassium cyanide is added to that of (I) with constant stirring. The white precipitate that is formed is filtered off and dried in the vacuum oven (3.2 parts). Elemental analysis of the product gave the following results (theoretical values in brackets): C 53.6 (53.9); H 3.0 (3.4); N 15.8 (15.7); S 16.0 (18.0). IR analysis showed -1 -1 3200 - 3450 cm-1 ; 1675 & 1415 -1 stretches at 3a00 - 3450 cm ; 1675 & 1415 cm ; 2165 cm & 730-750 cm indicative of -CONHal -SCN and an ortho disubstituted benzene ring. Example 2 Preparation of 5 chloro-2-thiocyanatobenzamide (5C2TB) (a) Preparation of 2 thiocyanato-5'-chlorobenzoic acid 11.2 parts of 5 chloroanthranilic acid, 2.61 parts of sodium hydroxide and 140 parts of water, are stirred to solution. 5.37 Parts of sodium nitrite dissolved in 70 parts of water are added. The whole is cooled to (50C. . 42.3 Parts of concentrated hydrochloric acid are added, with constant agitation. The temperature is held at < 5 C throughout the acid addition and for a further 30 minutes. The 'product is a solution of diazo 5chloroanthranilic acid. 18.87 parts of potassium thiocyanate with 7.92 parts of cuprous thiocyanate, in 280 parts of water1 are heated to 700C. The above diazo solution is then added, over a 30 minute period, maintaining a temperature of 700C. The whole is stirred for a further 30 minutes at the same temperature, cooled to room temperature and filtered. The filter cake is washed with 200 parts of water and pulled semi-dry at the pump. The filter cake is suspended in 220 parts of acetone, refluxed, filtered hot, to remove inorganic insolubles and cooled to room temperature. The solution is evaporated to 520 parts and cooled in a solid carbon dioxide/ acetone bath, yielding 5.48 parts of a brown-yellow solid. Analysis of solid gave the following results: (i) m.pt. 146-130C (ii) IR Spectrum broad band at 3250-2950 cm indicative of OH -1 a sharp peak at 2170 cm indicative of SCN a sharp peak at 1680 cm 1 C = O and peaks at 900 and 820 cm indicative of a tri-substituted benzene ring. (b) Preparation of 2 thiocyanato-5-chlorobenzoyl chloride 5.48 Parts of the product of (a) and 56.8 parts of dry ether are charged to a reaction vessel, and 6.39 parts of phosphorus pentachloride are added slowly. The mixture is stirred at room temperature for 18 hours, the ether is distilled off under high vacuum at (3COC, and the vacuum (o.6 mm Hg) is maintained for one hour. The resulting solid is extracted with 177.5 parts of dry ether, filtered to remove insoluble impurities, and cooled in solid carbon dioxide/acetone bath, until crystallisation commences. The crystals are filtered, dried over phosphorus pentoxide, in a dessicator, to yield 3.21 parts of a product hawing a melting point at 93-94 C. (c) Replacement of chlorine atom by amino group 0.83 Parts of product of (b) and 42.6 parts of dry ether are stirred and cooled in solid carbon dioxide/acetone bath. 9.607 Parts of ammonia dissolved in 7.1 parts dry ether is added in portions over a ten minute period. The whole is stirred with the above cooling for one hour and for a further hour at room temperature. The resulting suspension is filtered and the filter cake ?rashed with (2 x 7.0 parts) ether. The ether extracts are combined and evaporated to dryness to give a white solid which,is recrystallised twice from ethanol to yield a white crystalline product, (5 chloro 2 thiocyanato benzamide). The results of analysis are: (i) m.p. 239-2410C (ii) elemental analysis (theoretical values in brackets): C 45.0% (45.2); H 2.3% (2.3); N 12.6$ (13.2); S 14.7% (15.0). (iii) IR Spectrum -1 -1 Peaks at 3200-3450 cm , 1670 & 1415 cm indicative of CONH2 and at 2150 cm indicative of SCN. Example 3 Preparation of 2 thiocyanato N-methyl benzamide (2TNMB) (a) Preparation of 2(N-methyl carbamoyl)benzene thiosulphonate 10 Parts of N-methyl benzisothiazolone are added to 60 parts sodium bisulphite. The temperature is raised to 500C, with constant stirring, at which point all the benzisothiazolone is dissolved, the whole is maintained at 50 C for 15 minutes, cooled to 0 C and evaporated to dryness. The resulting solid is extracted twice with 100 parts of boiling methanol, filtered hot to remove inorganic impurities and evaporated to dryness to yield 15 parts of a white solid. -1 The IR spectrum showed bands at 3300 cm , 3100 cm 1640 cm and 1550 cm indicative of -CONH- and bands at 1210-1740 cm-1 and 1030 cm-1 indicative of -SO3Na. (b) Conversion of thiosulphonate group to thiocyanate group 2.69 Parts of the product of (a) are dissolved in 50 parts of water and a solution of 0.65 parts of potassium cyanide in 20 parts of water is added dropwise. A white precipitate forms. This is filtered off and dried to give 0.65 parts of 2 thiocyanato-N-methyl benzamide. The IR spectrum-showed peaks at 3200-3400 cm -1 of CO-NH-, 2615 cm-1 indicative of -SCN, and 7300-750 -1 indicative of CO-NE-, 2165 cm -1 indicative of -SCN, and 730-750cm indicative of an ortho disubstituted benzene ring. Example 4 Preparation of 2 thiocyanato-N-ethyl benzamide (2TNEB) (a) Preparation of sodium 2-(N-ethyl carbamoyl) benzene thio sulphonate 18.1 0irts of N-ethyl benzisothiazolone is suspended in 136 parts sodium bisulphite liquor (30%), the benzisothiazolone forming a top layer and the suspension is heated, with constant stirrings, at 32 C, when the reaction mass becomes homogeneous. The temperature is allowed to rise to 50 C, it is then cooled to 0 C. The solution is evaporated to dryness, under vacuum, to yield a white solid which is extracted twice with 100 parts of boiling methanol, and evaporated to dryness to yield 35.2 parts of a white solid. The IR spectrum showed bands at 33001 3100, 1640 and 1550 cm , indicative of -CONH- and at 1210, 1240 and 1030 cm indicative of -S03Na. (b) Conversion of thiosulphonate group to thiocyanate group 2.83 Parts of the product of (a) are dissolved in 20 parts of water, and 0.65 parts of potassium cyanide are dissolved in 10 parts of water. The potassium cyanide solution is added to the first solution with constant stirring. After a short time (s 5 mins) an oil separated as a: lower layer. Stirring is continued for a further 2 hours, after which the oil solidifies. The resultant white solid is filtered off and dried in a vacuum oven. Elemental analysis gave the following results (theoretical values in brackets): C 59.OSk (58.3); H 5.ZX (4.9); N 10.1% (13.6); S 14.7% (15.5). The IR spectrum showed the same characteristic peaks as the product of Example 9. Assessment of SamPles Assessment 1 - -In-vitro Antifungal and Antibacterial Activity of 2TB (ex 1), 5C2TB (Ex 2) and 2TNDB (Ex 3) 1% solution6 of each of these biocides in dimethylformamide were added to molten malt agar and to nutrient agar media to give in each case a final concentration in the test medium of 100 ppm. The test media were poured into petri dishes and allowed to cool before inoculation with test microorganisms. Nutrient agar was inoculated with three different test bacteria and malt agar was inoculated with five test fungi by means of an AM 80 automatic inoculator. The nutrient agar plates were examined for the presence or absence of bacterial growth after incubation at 370C for 24 hours and the malt agar dishes were examined for the presence or absence of fungal growth after incubation at 250C for 5 days. The results of the assessments are given in the following table(Table I): Table I EMI13.1 <tb> <SEP> Growth <SEP> of <tb> Biocide <tb> <SEP> Bacteria <SEP> Fungi <tb> <SEP> A <SEP> B <SEP> C <SEP> D <SEP> E <SEP> F <SEP> G <SEP> <SEP> H <SEP> <tb> Control <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> 2TB <SEP> + <SEP> - <SEP> <SEP> - <SEP> <SEP> - <SEP> <SEP> + <tb> 5C2TB <SEP> + <SEP> - <SEP> <SEP> - <SEP> * <SEP> + <SEP> <SEP> : : <SEP> <tb> 5B <SEP> + <SEP> + <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> <tb> + = Growth - = No growth (i.e. total inhibition) A = Pseudomonas aeruginosa D = Pullularia pullulans B = Escherichia coli E = Aspergillus niger C = Staphylococcus aureus F = Cladosporium sphaerospermum G = Altenaria tenuis (not tested *) G = Altenaria tenuis H = Chaetomium globosum Assessment 2 - Activity of 2T3 as an In-can Paint Preservative 2TB as a 1% solution in dimethylformamide was added to 50 g quantities of a styrene-acrylic emulsion paint to give final concentrations of 100 ppm and 200 ppm in the wet paint. The control contained no biocide but an equal amount of dimethylformamide to that present in the paint containing 200 ppm of biocide. Each paint was inoculated with 0.5 ml of a mixture of overnight broth cultures of 3 bacteria once per week for 2 weeks. Survivors were determined each week at 1 and 3 days after each inoculation by the decimal dilution method. The results of the assessment are given in the following table (Table II): Table II EMI14.1 <tb> <SEP> Survivors <tb> Sample <SEP> Week <SEP> 1 <SEP> Week <SEP> 2 <tb> <SEP> Day <SEP> 1 <SEP> Day <SEP> 3 <SEP> Day <SEP> 1 <SEP> Day <SEP> 3 <tb> 200 <SEP> ppm <SEP> 2TB <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10 <tb> 100 <SEP> ppm <SEP> 2TB <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10 <tb> Control <SEP> 8.o <SEP> x <SEP> 106 <SEP> 2.0 <SEP> x <SEP> 107 <SEP> 5.0 <SEP> x <SEP> 10 <SEP> 2.0 <SEP> x <SEP> 107 <tb> The bacteria used in the assessment were A and B from Assessment 1 and Enterobacter cloacae. The results demonstrate that, at a level of 100-200 ppm 2TB has a high activity as an in-can paint preservative. Assessment 3 - Activity of ZTB as a Paint Film Fungicide 2TB was added to a PVA-copolymer emulsion paint to give a concentration of 3000 ppm in the wet paint. The paint had the following composition: EMI14.2 <tb> Tap <SEP> water <SEP> 258 <SEP> g <tb> Ethylene <SEP> glycol <SEP> 10 <SEP> g <tb> Cellosolve <SEP> 7.5 <SEP> g <tb> Nopco <SEP> NDW <SEP> 1.5 <SEP> g <SEP> milibase <SEP> <SEP> 63.1% <SEP> <tb> <SEP> millbase <SEP> 63.1% <tb> Calgon <SEP> PT <SEP> (50% <SEP> solution) <SEP> 2 <SEP> g <tb> Cellofas <SEP> B50 <SEP> 5.5 <SEP> g <tb> China <SEP> Clay <SEP> Grade <SEP> D <SEP> 67.5 <SEP> g <tb> Tioxide <SEP> RCR <SEP> 280 <SEP> g <tb> <SEP> Total <SEP> 632 <SEP> g <tb> PVA copolymer emulsion (ICI x 935-6) 36. 0.880 ammonia 0.1% 0.3 g 2TB was added to 63 g millbase mixture and 50 g beads was added. The millbase- fungicide mixture was "red- devilled" for 35 minutes and 37 g of latex was added slowly , with hand stirring. The beads were sieved out of the emulsion paint and the latter was transferred into glass bottles which were tightly sealed and stored at 500C for 5 weeps. The control paint contained no added fungicide. 12"x6"xµ" Cedar panels were sprayed with one coat of Dulux* white wood primer and two coats of Dulux* Brilliant White undercoat. Two coats of the emulsion paint were a applied, by brush, to. one side of a panel, and 24 hours were allowed between each coat for drying. The backs and edges of the panels were painted with one coat of aluminium paint in order to seal the surfaces not coated with the emulsion paint. The painted panels were exposed in a Malayan jungle site and were examined regularly for the development of mould growth. The extent of contamination was recorded as the % of the panel covered by the mould growth.(Table III) (* Dulux is a trade mark belonging to Imperial Chemical Industries Limited.) Table III EMI16.1 <tb> Sample <SEP> Replicate <SEP> % <SEP> Area <SEP> Moulded <SEP> After <SEP> Exposure <tb> <SEP> Panels <SEP> Period <SEP> of: <SEP> (weeks) <tb> <SEP> 10 <SEP> 13 <SEP> 16 <SEP> 18 <SEP> 20 <SEP> 22 <SEP> <SEP> 24 <SEP> 26 <SEP> 28 <SEP> <tb> 2T13, <SEP> 3000 <SEP> ppm <SEP> I <SEP> O <SEP> 0 <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 10 <SEP> 10 <SEP> 10 <tb> <SEP> 2 <SEP> O <SEP> O <SEP> 5 <SEP> 5 <SEP> 10 <SEP> 15. <SEP> 15 <SEP> 20 <SEP> 20 <tb> Control <SEP> 1 <SEP> O <SEP> <SEP> 80 <SEP> 90 <SEP> 95 <SEP> 100 <SEP> - <SEP> <SEP> - <SEP> <SEP> - <SEP> <SEP> - <SEP> <tb> <SEP> 2 <SEP> <SEP> 85 <SEP> 95 <SEP> 100 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> The results indicate that 2TB possesses marked paint film fungicide activity. Assessment 4 - Antialgal Activity of 2TB Ankystrodesmus spirilliformis, Stichococcus bacillaris, and Chlamydomonas reinhardii were inoculated into one-litre batches of Difco algal broth and incubated at 18 C for 2 weeks in an illuminated incubator. The three cultures were then combined and the mixed culture was dispensed in 50 ml volumes into 100 ml sterile conical flasks. 2TB was added to each flask to give final concentrations of 5.0 and 10.0 ppm. The control consisted of the same algal mixture without the addition of biocide. The flasks were incubated up to 14 days in the illuminated incubator and were examined for the presence or absence of the green colouration of the algal cells. The retention of the green colour indicated that the algae remained viable, whereas discolouration of the solution indicated that the algae had been killed. The results are shown in Table IV. Table IV EMI17.1 <tb> <SEP> Survival <SEP> of <SEP> Algae <SEP> After <tb> <SEP> Sample <tb> <SEP> 3 <SEP> days <SEP> 7 <SEP> days <SEP> 14 <SEP> days <tb> 2TB, <SEP> 5.0 <SEP> ppm <SEP> + <SEP> + <SEP> + <tb> 2TB, <SEP> 10.0 <SEP> ppm <SEP> <SEP> - <SEP> - <SEP> - <SEP> <tb> Control <SEP> (no <SEP> biocide) <SEP> + <SEP> + <SEP> + <tb> The results demonstrate that 2TB has algicidal activity. Example 5 Preparation of 2-thiocyanato N-t-butylbenzamide (a) Preparation of 2(N-t-butylcarbamoyl)benzene thiosulphonate 8.28 Parts of N-t-butylbenzisothiazolone are suspended in 80 parts of sodium bisulphite liquor (40%), the benzisothiazolone forming a top layer. The suspension is heated, with constant stirring till at 630C the reaction mass becomes homogeneous. The temperature is held at 630C for a further 30 minutes. The solution is cooled to room temperature and 9.6 parts of a white solid crystalli.es out. IR spectrum shows band at 3300, 3100, 1640, 1550cm indicative of -CONH- and at 1210, 1240 and 1030cm indicative of -S03Na. (b) Conversion of thiosulphonate group to thiocyanate group 2.82 Parts of the product of (A) are suspended in 50 parts of water. This is heated, with continuous stirring to 400C, at which point the solid dissolves. Maintaining the above temperature, 0.65 part- of potassium cyanide dissolved in 10 parts of water is added, an immediate precipitate is formed. The reaction mass is cooled to room temperature, the solid filtered off and dried in the vacuum oven. Elemental analysis gives the following results: (Theoretical in brackets) C 61.5% (62.3); H 6.3% (4.8) Example 6 Preparation of 2-thiocyanato N-2-chloroethylbenzamide (2TN2CEB) (a) Preparation of 2(N-2-chloroethylcarbamoyl )benzenethio- sulphonate 2.14 Parts of N-2-chloroethylbenzisothiazolone are suspended in a solution of 4 parts of sodium bisulphite liquor (40$) and 50 parts of water. The reaction mass is heated with stirring to 5000. At this temperature the majority of solid is dissolved, leaving trace impurities. The liquors are decanted off from the impurities and allowed to cool to room temperature. As no solid precipitates from solution, the reaction mass is evaporated to dryness to yield a tacky solid. This is extracted with boiling ethanol, filtered at the boil to remove inrganics, cooled to room temperature and evaporated to dryness to yield 2.9 parts of a white powdery hygroscopic material. (b) Conversion of thiosulphate group to thiocyanate group 1.4 Parts of the product of (A) are dissolved in 20 parts of water and 0.286 part of potassium cyanide are dissolved in 10 parts of water. The potassium cyanide solution is added to the first solution with constant stirring. An immediate precipitate is formed. This is filtered off and dried at the pump. Elemental analysis gives the following results: C 50.1% (49.9); H 3.7% (3.7); N 10.8% (11.6); Cl 13.3% (14.8); S 14.0% (13.3). IR shows character similar to that of the product of Example 3. Example 7 Preparation of 2-thiocyanato-N-2-hydroxyethylbenzamjde (2TN2HEB) (a) Preparation of 2-(N-2-hydroxyethylcarbamoyl)benzene thiosulphonate Procedure as for Example 6 using 3.9 parts of N-2-hydroxyethylbenzisothiazolone and 40 parts of sodium bisulphite liquor (40%). Reaction mass heated to 500C, cooled. As no solid precipitates out, solution is evaporated dry to yield a white solid which is extracted with boiling ethanol, filtered hot to remove inorganics, cooled, and evaporated dry to yield 4.5 parts of a white powdery solid. IR spectrum 'shows bands typical of thiosulphonates of previous Examples. (b) Conversion of sulphonate group to thiocyanate group 1.5 Parts of the product from (a) are dissolved in 5 parts of water. 0.32 Part of potassium cyanide are dissolved in 2 parts of water. ' The potassium cyanide solution is added to that of the first solution with constant stirring. An immediate precipitate is formed. This is filtered off and dried in the vacuum oven. Elemental analysis gives the following results: C 52.5% (54.1); H 4.4% (4.5); N 11.8% (12.6); S 13.9 (14.4). IR spectrum shows same characteristic peaks as Example 3. Example 8 Preparation of 2-thiocyanato-N-allylbenzami de (2TNAB) (a) Preparation of 2 (N-allylcarbamoyl )benzenethiosulphonate 5.2 Parts of N-allylbenzisothiazolone are suspended in a solution of 9 parts of sodium bisulphite liquor (40%) and 50 parts uf water. The reaction mass is heated with stirring to 500C, at which point all solid is in solution, cooled to room temperature, and as no solid precipitates out, evaporated to dryness to yield a yellow solid. This solid is extracted with boiling ethanol, filtered hot to remove inorganics, cooled and evaporated dry to yield 5.1 parts of a yellow solid. 13 'spectrum shows bands typical of previous thiosulphonates. (b) Conversion of thiosulphonate group to thiocyanate group 1.47 Parts of the product from (a) are dissolved in 10 parts of water. 0.325 Part of potassium cyanide are dissolved in 5 parts of water. The solution of the product from (a) is cooled in an ice bath, and with constant stirring the solution of potassium cyanide is added to it. An immediate yellow solid is formed. The yellow solid suspended in water is extracted with 10 parts of chloroform, which is separated off from the upper aqueous layer, dried over anhydrous magnesium sulphate, filtered and evaporated to yieldzthe required product. The IR spectrum shows the same characteristic peaks as the product of Example 3. Example 9 Preparation of 2-thiocyanato-N-neopentylbenzamide (2TNNPB) (a) Preparation of 2-(N-neopentylcarbamoyl)bensenethio- sulphonate 4.4 Parts of N-neopentylbenzisothiazolone are suspended in a solution of 5 parts sodium bisulphite liquor (40%) and 50 parts of water. The reaction mass is heated with stirring to 90or. At this temperature the solution is a pale yellow, and a brown oil has formed on the-walls of the reaction vessel. The solution is decanted off from the oil, cooled to room temperature and evaporated dry to yield a yellow solid which was extracted with boiling ethanol, filtered hot to remove inorganics, cooled and evaporated to dryness to yield 3.65 parts of a yellow solid. The IR spectrum shows bands typical of previous thiosulphonates. (b) Conversion of thiosulphonate group to thiocyanate group 3.2 Parts of the product from (a) are dissolved in 10 parts of water. 0.65 Part of potassium cyanide are dissolved in 10 parts of water, with constart stirring, the solution of cyanide is added to the solution of (a). A solid is precipitated immediately; this is filtered off and dried in vacuum oven. Elemental analysis shows: C 65.7% (67.2); H 7.1% (6.7); N 11.1% (12.0); S 13.1% (13.7). The IR spectrum shows same characteristic peaks as the product of Example 3. Example 10 Preparation of 4-chloro-2-thiocyanatobenzamide (4C2TB) 10.4 Parts of anhydrous ammonia is dissolved in 50 parts of dried tetrahydrofuran held at -600C by means of a cooling bath. 4.6 Parts of 4-chloro-2-thiocyanato benzoic acid chloride in 50 parts of tetrahydrofuran is added slowly dropwise to the ammonia solution at -600C, and the mixture is allowed to rise to ambient temperature before standing for 12 hours. The resultant suspension is filtered to remove inorganic material, the filtrates reduced to dryness, and the residue purified by fractional recrystallisation from ethanol to give 0.8 g of 4-chloro-2-thiocyanatobenzamide m.p. 224-2260C. The I.R. spectrum shows the characteristic -SCN absorption at 2160 cm Example 11 Preparation of 3-carbonamido-2-thiocyanatopyridine (3C2TP) 4.19 Parts of 2-chioronicotinamide, 4.55 parts of potassium thiocyanate and 43 parts of glacial acetic acid are stirred at 40-440C for 24hours under an atmosphere of nitrogen. The solution is poured onto 200 g crushed ice and the product filtered, washed with water and dried. Yield 0.63 g, m.pt. 179-181 C. A small portion when re-crystallised from aqueous acetone yields pale yellow crystals, m.pt. 192-194 C. The I.R.spectrum contains a thiocyanate band at 2160 cm 1. Analysis: Found: C 47.0; H 2.6; N 22.9; S 17.0. Calculated for C7H5N30S: C 46.9; H 2.8; N 23.5; S 17.9. Example 12 Preparation of 3-thiocyanato-2-nanhthoic acid amide (3T2NA) 3.84 Parts of anhydrous ammonia is dissolved in 50 parts of dry ether held at -60 C by means of a cooling bath. 1.81 Parts of3-thiocyanato-2-naphthoic acid chloride in 50 parts of dry ether is added dropwise over a period of ten minutes. An exotçerm of 15 is observed before the reaction mixture is stirred at -60 C for 1 hour followed by 25 hours at ambient temperature. The suspension is filtered to remove ammonium chloride, the ether filtrates reduced to dryness and the product purified by fractional crystallisation from ethanol to give 0.44 g of 3-thiocyanato-2-naphthoic acid amide m.p. 176-178 C. The I.R. spectrum shows the characteristic -1 -SCN absorption at 2150 cm Example 13 Preparation of 2-Thiocyanato-N-cyclohexylbenzamide (2TNCHB) (a) Preparation of Sodium 2(N-cyclohexyl carbamoyl)benzene thiolsulphonate (2NCHCBTS) ¯N-Cyclohexyl benzisothiazolone (3.3 parts) is distilled in methanol (25 parts). To 'this solution, whilst stirring, is added sodium bisulphite liquor, 40%, (28 parts); an exotherm of 80C occurs (i.e. 21 " 29 C). The temperature is raised to 400C, to ensure complete reaction, cooled to room temperature, filtered, and evaporated to dryness. The resultant solid is extracted with methanol, inorganic material filtered off, and the methanol solution evaporated to yield a light brown solid sodium (N-cyclohexyl carbamoyl)benzene thiolsulphonate (3.8 parts). The IR spectrum has bands at 3370, 3060, 1620, 1530 -1 cm arising from the -corz- group and bands at 1210 and -1 1030 cm arising from the -sO3Na group. (b) Conversion of thiosulphonate group to a thiocyanate group The sodium (N-cyclohexyl carbamoyl)benzene thiolsulphonate (3.8 parts) is dissolved in water (50 parts) and methanol (10 parts). To this solution is added potassium cyanide (0.715 parts) dissolved in water (10 parts). The white precipitate formed is filtered off and dried in a vacuum oven, to yield a white powder 2-thiocyanato N-cyclohexyl benzamide (2.1 g.). The IR spectrum has a peak at 2160 cm 1 due to the -SCN group. Example 14 Preparation of 5-Methyl-2-ThiocyÅanatobenzamide (5M?TB) (a) Preparation of sodium 2-carbamoyl-5-methylbenzene thiolsuiphonate (2C4FETS) 5-Methyl-benzisothiazolone (2.05 parts) is suspended in sodium bisulphite liquor, 40% (24 parts). The temperature is raised, with constant stirring, to 600C at which point the benzisothiazolone has dissolved, leaving a small quantity of undissolved impurity. The solution is filtered hot to remove the impurity and cooled to room temperature. A white solid precipitates out. This is filtered, extracted with boiling methanol, filtered hot, and evaporated to dryness to yield a white solid, sodium 2-carbamoyl-5-methyl benzene thiolsulphonate (3.3 g.). The IR spectrum shows the same characteristic peaks and bands as the product of Example 13(a). (b) Conversion of thiosulphonate group to thiocyanate group The sodium 2-carbamoyl -5 -methyl benzene thiolsulphonate (2.5 parts) is dissolved in water (25 parts). To this solution is ded potassium cyanide (0.58 parts) dissolved in water (25 parts). The white precipitate formed is filtered off and dried in vacuum oven, yielding a white powder, 2-thiocyanato-5-methyl benzamide (6.8 parts). The IR spectrum shows the same characteristic peaks associated with the -SCN group as the product of Example 13. Example 15 Preparation of 2-Thiocyanato -N-p-chlorophenyl benzanide (2TNpCPB) (a) Preparation of sodium (N-4-chlorophenyl carbamoyl) benzene thiolsulphonate (2NpCEBTS) 4-Chlorophenyl benzisothiazolone (2.9 parts) is suspended in ethanol (50 parts). To this suspension is added sodium bisulphite liquor 40% (2.5 parts). A 5 deg ('19240C) exotherm occurs and the benzisothiazolone dissolves. The mixture is stirred for a'further 30 minutes, filtered to remove insoluble inorganic matter and evaporated to dryness, yielding a grey solid, sodium [N-(4-chloro phenyl carbamoyl))benzene thiolsulphonate (4.0 parts). The IR spectrum shows the same characteristic peaks as the product of Example 13(a). (b) Conversion of thiosulphonate group to thiocyanate group Sodium [N-(4-chlorophenyl carbamoyl) )benzene thiolsulphonate (3.65 parta) is dissolved in water (5 parts). To this solution is added potassium cyanide (0.65 parts) distilled in water (5 parts). The white precipitate formed, is filtered off and dried in the vacuum oven yielding a white powder, 2-thiocyanato N-4-chlorophenyl benzamide (1.1 parts). The IR spectrum shows the peak at 2160 cm characteristic of the -SCN group. Example 16 Preparation of 2-Thiocyanato-N-benzylbenzamide (2TNBB) (a) Preparation of sodium 2(N-benzyl carbamoyl)benzene thiolsulphonate (2BCBTS) N-benzyl benzisothiazolone (4.8 parts) is dissolved in methanol (50 parts). To this solution is added sodium bisulphite liquor (40%), (5 parts). An exotherm of 9 degrees (i.e. 21-300) occurs. The temperature is allowed to return to ambient and the solution is filtered, evaporated to dryness to yield a tacky solid which is extracted with ethanol (100 parts). The resulting solution is decanted from undissolved solid, evaporated to dryness yielding a white solid, sodium 2(N-benzyl carbamoyl)benzene thiolsulphonate (6(4 parts). The IR spectrum shows the same characteristic peaks and bands as the product of Example 15(a). (b) Conversion of thiosulphonate group to thiocyanate group The sodium 2(N-benzyl carbamoyl)benzene thiosulphonate (3.45 parts) is dissolved in water (5 parts). To this solution is added potassium cyanide (0.65 parts) dissolved in water (5 parts). The precipitate formed is filtered off and dried in a vacuum oven, yielding a white solid,2-thiocyanato N-oenzyl benzamide (0.9 parts). The IR spectrum shows the peak at 2160 cm 1 characteristic of the -SCN group. Assessment 5 - In-vitro Antifungal and Antibacterial Activity of the products of Examples 4 to 16 and of the Thiosulphonate Intermediates in the processes of Examples 13 to 16 The products of Examples 4 to 16 and the thiosulphonate intermediates described in Examples 13 to 16 are assessed according to the procedure describeil in Assessment 1 and the results are displayed in the following table (Table V): : Table V EMI27.1 <tb> <SEP> Growth <SEP> of <tb> Biocide <SEP> Bacteria <SEP> Fungi <tb> <SEP> A <SEP> B <SEP> C <SEP> D <SEP> F <SEP> G <SEP> H <tb> Control <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> 2TNtBB <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> -* <tb> 2TN2CEB <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> * <tb> 2TN2HEB <SEP> + <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> + <tb> 2TNAB <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> 2TNNPB <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> 2T4CB <SEP> + <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP> <tb> 2T3CP <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> + <tb> 3T2NAA <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> <RTI ID=27.4> 2TNCHB <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> * <SEP> <tb> 5M2TB <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> * <tb> 2TNpCPB <SEP> + <SEP> + <SEP> - <SEP> - <SEP> + <SEP> - <SEP> * <tb> 2TNBB <SEP> + <SEP> + <SEP> - <SEP> + <SEP> + <SEP> + <SEP> * <tb> 2NCECBTS <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> * <tb> 2C4MBTS <SEP> + <SEP> + <SEP> - <SEP> ¯ <SEP> + <SEP> - <SEP> * <SEP> - <SEP> <tb> 2NpCPBTS <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> * <SEP> <tb> 2NBCBTS <SEP> + <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> * <tb> The letters A to H have the meanings defined in Assessment 1. (*not tested against this fungus).

Claims: CILAIMS 1. A method for protecting a medium from attack by micro-organisms which comprises incorporating into the medium a biologically-active compound containing an aromatic ring on which are substituted, in ortho-relationship to one another, a thiocyanate group and a carbamoyl group. 2. A method according to Claim 1 wherein the compound has the formula: EMI28.1 Formula I wherein X is C, S, N or O Z is C, N or a direct link R is H, C1 to C20alkyl, cycloalkyl, aryl, aralkyl or C3 to C20alkenyl and R1 and R2 are each independently H, halo, nitro, cyano, thiocyanate, C1 to C16alkyl or C1 to C4alkoxy or R1 and + together with the benzene ring forum a fused ring system. 3. A medium which is susceptible to attack by microorganisms containing from 5 to 50,000 parts per million, by weight, of the compound defined in Claim 1 or Claim 2. 4. A biologically-active compound containing an aromatic ring on which are substituted, in ortho-relationship to one another, a thiocyanate group and a carbamoyl group, provided that where the compound comprises a single benzene ring, either the benzene ring or the carbamoyl group carries at least one substituent other than hydrogen. 5. A compound of the formula: EMI29.1 Formula I wherein X is C, S, N or O Z is C, N or a direct link R is H, C1 to C20alkyl, cycloalkyl, aryl, aralkyl or C to C20alkenyl and R1 and R2 are each independently H, halo, nitro, cyano, thiocyanate, C1 to C16alkyl or C1 to C4alkoxy or. R1 and 32 together with the benzene ring form a fused ring system, provided that at least one of R, RÚ and Rê is a group other than hydrogen. 6. A compound according to Claim 5 wherein the alkyl, aryl, aralkyl or alkenyl radical represented by 3 is substituted. 7. A compound according to Claim 5 or Claim 6 wherein the alkyl or alkoxy radicel represented by R1 or R2 is substituted. 8. A process for the preparation of a compound according to Claim 2 which comprises reacting a compound of the formula: EMI30.1 wherein'X, Z, R, R1 and R2 have the meanings defined in Claim 2 and M is an alkali metal or an ammonium ion with an alkali metal cyanide in an aqueous medium at a temperature from 0 to 100 C. 9. A compound of the formula: EMI30.2 wherein X, Z, R, R1 and Ra have the meanings defined in Claim 5 and M is an alkali metal or an ammonium ion. 10. A process for the preparation of a compound according to Claim 9 which comprises reacting a compound of the formula: EMI30.3 wherein X, Z, R, R1 and R2 have the meanings defined in Claim 5 with an alkali metal bisulphite in an aqueous or an alcoholic medium at a temperature from 0 to 1000C.

Inventors: BUCKLEY, ALAN JOHN
Applicants: IMPERIAL CHEMICAL INDUSTRIES PLC
Application year: 1980
Main classification: A01N47
IPC codes: C07D213, A01N47, C07C325, C07C331, C07C67, C09D5