(2-one (or thione)-1,3-dithiole 4,5-diyl) bis(thiocyanate) compounds, compositions containing the compounds and their use as an antimicrobial and marine antifouling agents
FIELD OF THE INVENTION 
     The present invention is directed to (2-one(or thione)-1,3-dithiole 4,5-diyl) bis(thiocyanate) compounds, compositions containing said compounds and their use as an antimicrobial and marine antifouling agents. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to (2-one(or thione)-1,3-dithiole 4,5-diyl) bis(thiocyanate) compounds corresponding to the formula: ##STR1## wherein Z is oxygen or sulfur. 
     The present invention is also directed to antimicrobial compositions comprising an inert diluent in admixture with an antimicrobially-effective amount of a (2-one (or thione)-1,3-dithiole 4,5-diyl)bis(thiocyanate) compound. 
     The present invention is further directed to a method for inhibiting microorganisms in a microbial habitat comprising contacting said microbial habitat with a composition containing an antimicrobially-effective amount of said compounds. 
     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 the growth of said marine organisms on said surfaces. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The (2-one(or thione)-1,3-dithiole 4,5-diyl) bis(thiocyanate) of the present invention can be prepared by the reaction, in a solvent, of a 4,5-dimercapto-1,3-dithiole-2-one (or thione) metal salt corresponding to the formula ##STR2## wherein M represents cesium, lithium, potassium, sodium or zinc, with a cyanogen halide such as cyanogen bromide, cyanogen chloride, cyanogen fluoride or cyanogen iodide. 
     Representative solvents which can be employed in the present process are polar solvents and include among others, water, acetone, ethanol, dimethylformamide and dimethylsulfoxide. 
     The reaction is typically carried out at room temperature under a nitrogen atmosphere. 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. The reaction is allowed to continue over a period of from about ten minutes to about 20 hours. The reaction consumes the reactants in the ratio of two mole equivalent of the halide reactant per mole of the thione reactant. To insure the completion of the reaction, an excess of the halide reactant is normally employed. 
     After the reaction is complete, the product normally precipitates out and is recovered by filtration. To insure product precipitation, a slight amount of water and/or methanol can be added to the reaction mixture. The recovered product is washed with water or methanol and dried. 
     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. 
     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 use of an excess of one reactant, the use of a catalyst, the use of temperatures slightly higher than room temperature and/or high speed mixing and other such conventional changes are within the scope of the present invention. 
     The structure identity of the compound was confirmed by proton nuclear magnetic resonance spectroscopy ( 1  H NMR), recorded at 300 MHz; carbon nuclear magnetic resonance spectroscopy ( 13  C NMR) recorded at 75 MHz; infrared spectroscopy (IR) and mass spectrometry (MS). The reaction is conducted under a positive pressure of nitrogen. 
    
    
     EXAMPLE I 
     (2-Thione-1,3-dithiole 4,5-diyl)bis(thio)-bis(thiocyanate) ##STR3## 
     A mixture of 1.15 grams(g) (2.8 mmol) of disodium dithiolate and 0.6 grams(g) (5.6 mmol) of cyanogen bromide in 15 mL of dimethylformamide was reacted at room temperature for 1.0 hour. The reaction mixture was filtered to give 0.46 g of the title compound as a yellow powder melting at 129°-132° C. in a yield of 66 percent of theoretical. MS (EI) m/z 248 (M+), 250 (M+2), 223, 190, 172, 146, 114; C 5  N 2  S 5  requires 248. Upon analysis, the product was found to have carbon, hydrogen and nitrogen contents of 24.94, 0.02 and 10.25 percent respectively, as compared with the theoretical contents of 24.18, 0.00 and 11.28 percent, respectively, as calculated for the above-named structure. 
     Preparation of Starting Materials 
     The 4,5-dimercapto-1,3-dithiole-2-one (or thione) metal salt corresponding to the formula ##STR4## wherein M and Z are as hereinabove defined and employed as starting materials herein are well known compounds and can be prepared as taught in the literature. 
     The cyanogen halides employed as starting materials are well known compounds and can either be obtained commercially or prepared as taught in the literature. 
     Antimicrobial Activity 
     The compound of this invention is useful as an antimicrobial additive, and can be added to industrial products such as paints, inks, adhesives, soaps, cutting oils, textiles, and paper and pigment slurries and to styrene-butadiene latexes used for paper coatings. 
     The compound is also useful as an antimicrobial additive 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. 
     The present invention is also directed to a method for inhibiting microorganisms which comprises contacting said microorganisms or habitat thereof with a composition containing an antimicrobially effective amount of (2-one (or thione)-1,3-dithiole 4,5-diyl) bis(thio)-bis(thiocyanate). 
     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 &#34;microorganism&#34; is meant to refer to bacteria, fungi, viruses, algae, subvital agents and protozoa. 
     As used herein, the term &#34;antimicrobially-effective amount&#34; refers to that amount of the compound, or of a composition comprising such compound, of this invention needed to exhibit inhibition of selected microorganisms. Typically, this amount varies from providing about 1 part per million (ppm) to about 5,000 ppm by weight of the compound to a microbial habitat being contacted with the compound. Such amounts vary depending upon the particular microorganism treated. Also, the exact concentration of the compound 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 about 1 ppm to about 500 ppm, more preferably from about 1 ppm to about 50 ppm by weight, of a microbial habitat. 
     The term &#34;microbial habitat&#34; 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 &#34;inhibition&#34;, &#34;inhibit&#34; or &#34;inhibiting&#34; 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 compound of the present invention is demonstrated by the following techniques. 
     The antimicrobial activity of the compound is set forth as the minimum inhibitory concentration (MIC) for the active compound and is determined for nine (9) bacteria, using nutrient agar, and seven (7) yeast and fungi, using malt yeast agar. This determination is conducted using a one percent solution of the test compound 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 0.04M 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 ×200 mm test tubes, capped and autoclaved for 15 minutes at 115° C. 
     The test 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 therein. The contents of the test tubes are then transferred to respective petri 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 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 10 8  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&#39;s Replicator. For each microbial suspension, 0.3 mL was used to fill three wells (i.e., three wells of 0.3 mL each) so that for the nine different bacteria, 27 wells are filled. The Steer&#39;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 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 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 to 9 parts sterile saline). Aliquots of these dilutions are placed in individual wells of Steer&#39;s Replicator and petri plates inoculated as previously described. The petri plates are incubated at 30° C. and read after 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                                                     
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Organisms used in the Minimum                                             
Inhibitory Concentration Test                                             
Organism              ATCC No.                                            
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Bacteria                                                                  
Bacillus subtilis (Bs)                                                    
                       8473                                               
Enterobacter aerogenes (Ea)                                               
                      13048                                               
Escherichia coli (Ec) 11229                                               
Klebsiella pneumonias (Kp)                                                
                       8308                                               
Proteus vulgaris (Pv)  881                                                
Pseudomonas aeruginosa (Pa)                                               
                      10145                                               
Pseudomonas aeruginosa (PRD-10)                                           
                      15442                                               
Salmonella choleraesuis (Sc)                                              
                      10708                                               
Staphylococcus aureus (Sa)                                                
                       6538                                               
Yeast/Fungi                                                               
Aspergillus niger (An)                                                    
                      16404                                               
Candida albicans (Ca) 10231                                               
Penicillium chrysogenum (Pc)                                              
                       9480                                               
Saccharomyces cerevisiae (Sc)                                             
                       4105                                               
Trichoderma viride (Tv)                                                   
                       8678                                               
Aureobasidium pullulan (Ap)                                               
                      16622                                               
Fusarium oxysporum (Fo)                                                   
                      48112                                               
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     In Tables II and III, the MIC values of the compound of the present invention as compared to the MIC of a standard commercial preservative (with 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride as the active agent, and referred to in Tables II and III as &#34;STANDARD&#34;) are set forth for the bacteria organisms and yeast/fungi organisms which are listed in Table I. 
     
                                           TABLE II                                
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Minimum Inhibitory Concentrations for Test Compounds                      
in Bacteria Species (in ppm)                                              
Compound                                                                  
        ORGANISMS                                                         
(Example No.)                                                             
        Bs   Ea   Ec   Kp   Pv   PRD  Pa   Sc   Sa                        
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STANDARD                                                                  
pH 6.8  &lt;10  100    50  25   50  &gt;500 &gt;500   50  25                       
pH 8.2  250  500  &gt;500 500  500  &gt;500 &gt;500 &gt;500 500                       
(I)                                                                       
pH 6.8  100  250   250 250  250  &gt;500 &gt;500  250 250                       
pH 8.2  &gt;500 &gt;500 &gt;500 &gt;500 &gt;500 &gt;500 &gt;500 &gt;500 &gt;500                      
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                                           TABLE III                               
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Minimum Inhibitory Concentrations for Test                                
Compounds in Yeast/Fungi Species (in ppm)                                 
COMPOUND  ORGANISMS                                                       
EXAMPLE NO.                                                               
          An  Ca  Pc  Sc Tv  Ap  Fo                                       
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STANDARD  &gt;500                                                            
              &gt;500                                                        
                  &gt;500                                                    
                      500                                                 
                         &gt;500                                             
                             &gt;500                                         
                                 &gt;500                                     
I          &lt;10                                                            
                25                                                        
                    25                                                    
                       25                                                 
                           50                                             
                              &lt;10                                         
                                   50                                     
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     Marine Antifouling Activity 
     The present invention is also directed to a method for inhibiting marine organisms. The term &#34;marine organisms&#34; is meant to include marine animals, such as barnacles, serpulid, bryozoa, oysters and hydroids, and marine plants, 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 antifouling effective amount of the compound of this invention. 
     As appreciated by those skilled in the art, there may be some variation in marine antifouling potency and spectrum of marine antifouling activity dependent on various factors including the specific materials with which the compound is formulated. 
     As used herein, the term &#34;marine antifouling effective amount&#34; refers to that amount of the compound of this invention needed to exhibit inhibition of selected marine organisms. Typically, this amount varies from providing about 1 weight percent to about 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 marine organism to be treated. Also, the exact concentration of the compound 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 binders are formulated as latexes or emulsions. In a paint composition, the compound of the present invention is preferably used in an amount from about 1 to about 30 weight percent and, most preferably, from about 10 to about 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 substantially free from build-up of marine organisms for periods ranging from about 3 to about 12 months, depending upon the concentration of the compound and the thickness of the applied coating or film. 
     The term &#34;a surface exposed to a marine environment&#34; refers to a surface where a marine organism naturally or normally lives or grows. Typically, such a surface will be an area 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 0.381×10 -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. 
     The candidate marine antifoulant compound (1.0 g) is stirred into a resinous latex binder (9.0 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 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 antifoulant compound is attached over a white background 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 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 (2-Thione-1,3-dithiole 4,5-diyl) bis(thio)-bis-methyl thiocyanate is set forth, as well as the ratings for control panels (with no marine antifouling compound and referred to as &#34;Control&#34;). 
     
                       TABLE IV                                                    
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Marine Antifouling Rating for Test Compounds                              
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             Marine Antifouling Ratings                                   
             Top Panel      Bottom Panel                                  
             at indicated   at indicated                                  
             time in weeks  time in weeks                                 
Test Compound  6       10       6     10                                  
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(2-Thione-1,3-dithiole                                                    
               7       2        3     2                                   
4,5-diyl) bis(thio)-                                                      
bis(thiocyanate)                                                          
Control        6       3        0     0                                   
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