CA2073458A1 - Composition and use of substituted 3-thio-2-propynenitriles as an industrial antimicrobial - Google Patents

Abstract

Substituted 3-thio-2-propynenitriles are prepared which correspond to the formula R-S-C=C-C=N, wherein R is an alkyl, cyclic alkyl, aryl, or heterocyclo group. These compounds have been found to exhibit a high degree of antimicrobial activity in industrial and commercial applications and compositions containing these compounds are so employed.

Description

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Wo91/l1503 PCT/US9~,~Q0262 , ,~: , COMPOSITION AND USE OF SUBSTITUTED

AS AN INDUSTRIAL ANTIMICROBIAL

The field of this invention is novel compounds which are useful as antimicrobials.
V.S. Patents 4,172,892 and 4,388,314 disclose an acid of the formula: :
HOOCCH2-S-C~C-C~N
10 which is used as a reactant in a process to produce a ~ :
compound of the formula:

15 N a C-C = C-S-CH2-c-N~F~ S

O ~CH2-S-B

wherein Xis a free or esterified carboxy group and B is a substituted tetrazolyl or thiadiazolyl radical or a ~.
heterobicyclic ring. This compound is taught to be wos~ o3 ~ ~ 3 ~ 5 8 PCT/US91/00262 useful in pharmaceutieal and veterinary compositions, as well as antibacterial activities.
PCT International Publication Number W0 89/07890, published 8 September, 1989, by Harold A. Brandman et al., discloses an ~-halo-~-(substituted)thio-acrylonitrile of the formula:

/ CN
RS-CH=C \
X

wherein X is a halogen and R is a lower alkyl, aryl, aralkyl, heterocyclo, or a thiocarbonyl group. This compound is taught to be useful as an effective antimicrobial agent.
The desirability of identifying or discovering new antimicrobial agents is widely recognized for several reasons. These include the development of microbe strains resistant to known antimicrobials, the occurrence of undesirable interactions of certain known antimicrobials with the medium or product in which the antimicrobial is used, and high toxicity of certain known antimicrobials to certain non-target organisms such as mammals.
The present invention solves this problem by 3 di~closing a new compound which may be employed as an antimicrobial.
The present invention is a compound corresponding to the formula:

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( ~-S-C-C-C=N
wherein R is an alkyl, cyclic alkyl, aryl, or heterocyclo group. The invention also includes a method Or making such substituted 3-thio-2-propynenitriles of the same formula, and compositions containing said compounds and the use of such compositions as antimicrobials in industrial or commercial uses.
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 i9 some compound-to-compound variation in antimicrobial potency and spectrum of antimicrobial activity.
The present invention is also directed to a process of using such substituted 3-thio-2-propy-nenitriles of the same formula as above, as an antimicrobial agent. This process is a method for inhibiting microorganisms, particularly bacteria, fungi~
and algae which comprises contacting said microorganisms .:~ ... . . :

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or habitat thereof with an effective amount of the co~pound 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 organic solvents like glycols, alcohols 7 or acetone. They may also be added alone or in combination with other preservatives.
In the present specification and claims, the term "alkyl" is employed to designate straight chain and branched chain alkyls. Such alkyls may be with or without substituents, such as cyclic alkyl, aryl, alkoxy or halogen. Preferably, the term "alkyl" is employed to designate straight chain alkyls of 1 to 18 carbon atoms and branched chain alkyls of 3 to 18 carbon atoms. Most preferably, the term "alkyl" is employed to designate straight chain alkyls of 1 to 12 carbon atoms, suah as methyl, ethyl, propyl, butyl, decyl, or dodecyl and branched chain alkyls of 3 to 12 carbon atoms, such as isopropyl or tertiary butyl.
In the present specification and claims, the term "cyclic alkyl" is employed to designate a closed-ring alkyl structure. Such cyclic alkyls may be with or without substituents, such as alkyl, aryl, alkoxy or halogen. Preferably, the term "cyclic alkyl"
is employed to designate cyclic alkyls of 3 to 8 carbon atoms. Most preferably, the term "cyclic alkyl" is employed to designate cyclic alkyls of 3 to 6 carbon atoms, such as cyclopentyl or cyclohexyl.
In the present specification and claims, the term "aryl" is employed to designate groups which have ' ............... . . .' . , '. ' .
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the ring structure characteristic of benzene, wherein the ring may be with or without substituents such as alkyl, cyclic alkyl, alkoxy, or halogen. The aryl ring may also be a fused ring, wherein the ring may have one or more of its sides ln common with another ring.
Preferably, the aryl ring has no more than three substituents. Most preferably, the aryl is phenyl, naphthyl, or chlorophenyl.
In the present specification and claims, the term "heterocyclo" is employed to designate a closed-ring structure containing at least one ring carbon, in which one or more of the atoms in the ring is an element other than carbon. Such heterocyclos may be with or without substituents, such as alkyl, cyclic alkyl, aryl, alkoxy, or halogen. The heterocyclo ring may also be a fused ring, wherein the ring may have one or more of its sides in common with another ring.
Preferably, the closed-ring structure will con~ist of 5 or 6 atoms. PrePerably, the non-carbon ring atom or atoms will be nitrogen, oxygen or sùlfur. Preferably, the ring has no more than three substituents. Most preferably, the heterocyclo is thiazolyl, triazolyl 9 imidazolyl, or pyrimidyl.
As used herein, the term "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 organisms. Typically, this 3 amount varies from 1 part per million (ppm) to 5000 ppm by weight. Such amounts vary depending upon the particular compound tested and organi~m 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 ..
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components of the formulation. In such formulations, the compounds of this invention may be added as a liquid concentrate or diluted with additional liquid to produee the ultimate treating composition, wherein the liquid .
could be water or an organic solvent such as glycols, alcohols, or acetone.
The terms "inhibition", "inhibit" or "inhibiting" refer to suppression, control, stasis, kill or any other interference with the normal life processes of microorganisms that is adverse to such microorganisms.
As used herein, the term "appropriately substituted 2-chloro-3-thio-2-propenenitrile precursor"
refers to a 2-chloro-3-thio-2-propenenitrile of the formula:

R-s-cH=C/
\ C--N

wherein R is an alkyl, cyclic alkyl, aryl, or heterocyclo group, which is used as a precursor to produce a like-substituted 3-thio-2-propynenitrile. As used herein, the term "like-substituted 3-thio-2-propynenitrile" refers to a 3 3-thio-2-propynenitrile of the formula:
R-S-CsC-CsN
wherein R is an alkyl, cyclic alkyl, aryl, or heterocyclo group, but wherein R is identical to the R
of the appropriately substituted 2-chloro-3-thio-- -. . . . . .

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-2-propenenitrile used as a precursor to produce the like-substituted 3-thio-2-propynenitrile. Thus, for example, 2-chloro-3-methylthio-2-propenenitrile is an appropriately substituted 2-chloro-3-thio--2-propenenitrile which is used as a precursor to produce 3-methylthio-2-propynenitrile, the like-substituted 3-thio-2-propynenitrile.
The compounds of the present invention can be prepared by the reaction of an appropriately substituted 2-chloro-3-thio-2-propenenitrile precursor with basic aqueous solution (such as sodium hydroxide solution).
In carrying out this reaction, the appropriately substituted 2-chloro-3-thio-2-propenenitrile precursor and the basic aqueous solution are mixed together in substantially equimolar amounts. The general reaction scheme is as follows:

/ Cl Basic R-S-CH=C\ Solution R-S-C-C-C--N
C=N

A preferred method of preparing the compounds of the present invention is to carry out the dehydrochlorination of the appropriately substituted 2-chloro-3-thio-2 propenenitrile precur~or at a temperature below ambient, in the presence of water and 3o an inert, water-miscible solvent such as tetrahydrofuran, dioxane, isopropanol, polyglycols and their ethers, or dimethylformamide, with the subsequent addition of a known Lewis base such as an alkaline earth metal hydroxide. As used herein, "Lewis base" refers to compounds that form a covalent bond by donating a pair .

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of electrons, with neutralization resulting from a reaction between the base and an acid with formation of a covalent bond.
The reaction rate of this preferred method of preparing the compounds of the present invention is conveniently controlled by the rate of base addition coupled with external cooling. Room temperature, however, may be used as the starting reaction temperature to Sncrease the reaction rate. The reaction may also be accelerated by increasing the amount of inert, water-miscible solvent. An increase in the amount of inert, water-miscible solvent also makes the reaction mixture more homogeneous.
Advantages of using this preferred method of preparing the compounds of the preqent invention lnclude mild reaction conditions, a high yield reaction, relatively inexpensive reagenta, and a short reaction time. In addition, by using an inert, water-miscible solvent, a solvent extraction step can be eliminated because a desired product may be directly formulated as compared to a reaction process that uses a water--immiscible solvent to isolate the desired product. The reaction yield of this preferred method is also sufficiently high such that purification of a desired final product may not be required.
Tetraethylene glycol, for example, is a common formulating solvent for paints, pigment slurries, latexes, and metal working fluids. By uqing tetraethylene glycol as the inert, water-miqcible solvent in the dehydrochlorination reaction, a desired composition may be directly formulated which could be , .

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-g i used directly into a paint, pigment slurry, latex, or metal working fluid product.
Svnthesls of Intermediate Appropriately Substituted -Chloro-3-Thio-2-ProPenenitrile The synthesis of the appropriately ~ubstituted 2-chloro-3-thio-2-propenenitrile precursor begins with the chlorination of acrylonitrile to form 2,2,3-trichloropropionitrile. This chlorination is straightforward and is described in the art, such as in 10 N. C. Lorette, "The Addition of Chlorine to -Acrylonitrile", J. Or~. Chem., Vol. 26, pp. 2324-2327, 1960. Overall yields of over 90 percent based on acrylonitrile are achievable.
Dehydrochlorination of 2,2,3-trichloropropio-nitrile yields an isomeric mixture of 2,3-dichloro-acrylonitrile. This dehydrochlorination can be carried out by heating the 2,2,3-trichloropropionitrile in the presence Or a catalyst with yields of ôO to 100 percent.
Known catalysts include, but are not limited to, organic bases such as pyridine, polyvinylpyridine, and their hydrochloride salts and phase transfer catalysts such as tetraphenylphosphonium chloride, tetrabutylammonium chloride and ion exchange resins. Purification of the 2,3-dichloroacrylonitrile prior to subsequent reaction is optional.
The 2,3-dichloroacrylonitrile reacts with an alkaline earth metal salt of an appropriate mercaptan to 3 form the appropriately substituted 2-chloro-3-thio--2-propenenitrile precursor in alkanols or aprotic solvents. The reaction temperature, stoichiometries~
and mode of addition are important to obtain acceptable . .
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isolated yields (greater than 85 percent from 2,3-dichloroacrylonitrile).
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.
Example 1 - Preparation of 3-Methylthio--2-Propynenitrile Water (95 ml) is added to a 250 ml roundbottom flask equipped with an addition funnel, condenser, thermometer, pH probe and magnetic stirring bar.
2-Chloro-3-methylthio-2-propenenitrile (2.64 g, 97 percent purity) is then added. The head space of the reactor is padded with nitrogen.
An aqueous NaOH solution is produced by mixing lN NaOH (22.5 ml) with water (40 ml). This NaOH
solution is added to the addition funnel.
The aqueous NaOH ~olution is added to the flask over a 20-hour period while maintaining the solution at room temperature or below. A temperature ~etween zero and 5C is preferred.
The reaction mixture is cooled to 5C.
Dichloromethane (30 ml) is added to the reaction 3 mixture. The two-phase system is agitated and then tran~ferred to a separatory funnel. The dichloromethane phase is removed and the dichloromethane is vacuum distilled off. The remaining material weighs 1.70 g.
Ga~ chromatographic analysis (GC) of the residue reveals . ~ , . .

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.,,, --1 1--71 percent by area 3-methylthio-2-propynenitrile. A
calculated overall yield of 64 percent is achieved.
The crude reaction product is purified by column chromatography yielding 3-methylthio--2-propynenitrile in greater than 99 percent purity (by GC). The structure identity is confirmed by proton (1H) and carbon nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR) and gas chromatography/mass spectrometry (GC/MS).
Example 1A - Preparation of 3-Methylthio--2-Propynenitrile Using a Preferred Reaction Procedure Into a 1000 ml reaction flask is charged 2-chloro-3-methylthio-2-propenenitrile (150.4 g;
1.12 moles), tetrahydrofuran (230 ml) and water (180 ml) and then 4N aqueous sodium hydroxide solution (267 ml, 1.06 moles) is slowly added in at zero degree Celsius with stirring at such a rate so as to maintain the reaction pH below 11. The mixture becomes dark brown.
Progress of the reaction is~monitored by GC. The resultant mixture is stirred at room temperature for 8 hours and then diluted with 150 ml of saturated aqueous sodium chloride solution. The product is extracted twice with 150 ml portions of dichloromethane.
The dichloromethane extract is dried over anhydrou~
sodium sulfate, filtered and concentrated on a rotary evaporator to yield 106.8 g of dark brown oil. The dark brown oil is purified by fractional distillation to give 88 g of 3-methylthio-2-propynenitrile as a colorless ; clear oil (yield, 81 percent) which is greater than 99.5 percent pure by GC. It is further characterized by GC/MS, IR and 1H NMR.

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-12- ?, Example 2 - Preparation of 3-Ethylthio-2-Propvnenitrile CH3-CH2-S-C a C-CN
i Into a 250 ml reaction flask is charged 5 2-chloro-3-ethylthio-2-propenenitrile (8.2 g;
0.055 moles), tetrahydrofuran (15 ml) and water (50 ml) and then lN aqueous ~odium hydroxide solution (55.6 ml 9 0.055 moles) is slowly added in at zero degree Celsius with stirring at such a rate so as to maintain the 10 reaction pH below 11. The mixture becomes dark brown.
Progress of the reaction is monitored by GC. The resultant mixture is stirred at room temperature for ~ :
8 hours and then diluted with 150 ml of saturated 15 aqueous sodium chloride solution. The product is extracted twice with 100 ml portions of dichloromethane.
The dichloromethane extract is dried over anhydrous ~odium sulfate, filtered and concentrated on a rotary evaporator to yield 6.8 g of dark brown oil. The dark ?
20 brown oil i~ purified by silica gel flash column chromatography to give 4.0 g of 3-ethylthio--2-propynenitrile as a colorless clear oil (yield, 66 percent) which is greater than 99 percent pure -by GC
It is further characterized by GC/MS, IR and 1H NMR.

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t~ 2 ~ 7 3~ ~ 8 p~ Jssnf~ fi2 , ~ , Example 3 - Preparation of 3-t-ButYlthio--2-Propynenitrile H3C ~ - S-C~C-CN

Into a 250 ml reaction flask is added 2-chloro-3-t-butylthio-2-propenenitrile (1.8 g, 0.01 moles), tetrahydrofuran (35 ml), and water (35 ml).
The mixture ~s stirred at zero degree Celsius until the solid is completely dissolved and then aqueous sodium hydroxide solution (10 ml 1N NaOH, 0.010 moles, diluted with 50 ml water) is added slowly with stirring at such a rate so as to maintain the reaction pH below 11. The progress of the reaction is monitored by GC. The resultant reaction mixture is stirred at zero degree Celsius for three hours followed by one hour at room temperature and then diluted with 50 ml of saturated NaCl solution. The product is extracted twice with i 100 ml portions of dichloromethane. The combined dichloromethane extract is dried over anhydrous sodium sulfate, filtered and the filtrate is evaporated under vacuum to give 1.5 g of colorless oil. This oil is purified by silica gel column chromatography to yield ; 0.86 g 3-t-butylthio-2-propynenitrile (yield 61 percent) which is greater than 99 percent pure by GC. It is further characterized by GC/MS and IR.

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W091/llS03 ,'!'' 20;7~g~8 PCT/1]59~/00262 Example 4 - Preparation of 3-Cyclopentylthio--2-Propynenitrile I

Into a 250 ml reaction flask is added 2-chloro-3-cyclopentylthio-2-propenenitrile (4.0 g, 0.026 moles), tetrahydrofuran (40 ml ) and water (40 ml) and then aqueous sodium hydroxide solution (27 ml 1N
NaOH, 0.027 moles, diluted with 80 ml water) is added at zero degree Celsius slowly with stirring at such a rate so as to maintain the reaction pH below 11. The progres~ of the reaction is monitored by GC. The resulting mixture is stirred at room temperature for 4 hours and then diluted with 100 ml of saturated aqueous NaCl solution. The product is extracted twice With 100 ml portions of dichloromethane. The combined dichloromethane eXtraCt is dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under vacuum to give 4.0 g Of brown oil. ThiS oil is purified by silica gel column chromatography to yield 2.2 g of 3-cyclopentylthio-2-propynenitrile (yield 69 percent) which is greater than 99 percent pure by GC. It is characterized by GC/MS, IR and 1H NMR.
Example 5 - Preparation of 3-(4-Chlorophenyl)Thio-- -2-Pro~ynenitrile Cl~S-C--C-CN , ~, :~ - . . .

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Into a 250 ml reaction flask is added 2-chloro-3-(4-chlorophenyl)thio-2-propynenitrile (4.0 g, 0.017 moles), tetrahydrofuran (40 ml) and water (40 ml).
The mixture iY stirred at zero degree Celsius until the solid is completely dissolved and then an aqueous ~odium hydroxide solution (45 ml, 0.016 moles) is added slowly with stirring at ~uch a rate so as to maintain the reaction pH below 11. The progress of the reaction is monitored by GC. The resultant reaction mixture is stirred at zero degree Celsius for 30 minutes and then diluted with 100 ml of saturated aqueous NaCl solution.
The product is extracted twice with 100 ml portions of dichloromethane. The combined dichloromethane extract is dried over anhydrous sodium sulfate, filtered and the filtrate is evaporated under vacuum to give 2.9 g of white solid. This solid is purified by silica gel column chromatography to yield o.8 g 3-(4-chlorophenyl)-thio-2-propynenitrile as colorless shiny flakes (yield 25 percent) which is greater than 99 percent pure by GC.
It is further characterized by GC/MS and IR.
Example 6 - Preparation of 3-(2-Pyrimidyl)Thio--2-PropYnenitrile ~-- N
~ )--S-C a C-CN

Into a 250 ml reaction flask is added 2-chloro-3-(2-pyrimidyl)thio-2-propenenitrile (1.5 g, 0.007 moles), tetrahydrofuran (35 ml) and water (35 ml).
The mixture iY stirred at zero degree Cel~ius until ~he solid i~ completely di~solved and then aqueous sodium hydroxide solution (7.5 ml 1N NaOH, 0.0075 moles, ., ~, ' ; , , .~:

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diluted with 50 ml water) is added slowly with stirring at, such a rate so as to maintain the reaction pH below 11. The resultant reddish brown solution is stirred at zero degree Celsius for three hours followed by one hour at room temperature and then diluted with 50 ml of saturated aqueous NaCl solution. The product is extracted twice with 100 ml portions of dichloromethane.
The combined dichloromethane extract is dried over anhydrous sodium sulPate, filtered and the filtrate is evaporated under vacuum to give 0.~ g of pale yellow solid (yield 40 percent, purity 90 percent by GC/MS) which is characterized by GC/MS and lH NMR.
Example 7 - Preparation of 3-Dec~lthio-2-ProPvnenitrile CH3-(CH2)9-S-C a C-CN

Into a 50 ml reaction flask is charged 2-chloro-3-decylthio-2-propenenitrile (1.0 g, 0.0038 moles) and 50 ml of water. The mixture is stirred at room temperature for 10 minutes and then 0~5N
aqueous sodium hydroxide (7.70 ml) solution is added slowly. The resultant mixture is heated to 80C and held at that temperature for 16 hours. The mixture is then cooled to room temperature and the product is recovered by extraction using two 50 ml portions of dichloromethane. The combined dichloromethane extracts are dried over anhydrous Na2S04, filtered and concentrated on a rotary evaporator to yield 0.66 g of brown oil. This oil is purified by silica gel column chromatography to give 0.25 g of 3-decylthio--2-propynenitrile (yield 29 percent) which is 80 percent pure by GC/MS.

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-l7-Example 8 - Preparation of 3-Dodecylthio- --2-Propynenitrile CH3-(CH2)l1-S-C=C-CN

Into a 50 ml reaction flask is charged 2-chloro-3-dodecylthio-2-propenenitrile (1.7 g, 0.0059 moles), 30 ml of dichloromethane and 50 ml of water. The mixture is stirred at room temperature for lO minutes and then 0.5N aqueous sodium hydroxide (7.7 ml, 0.0039 moles) solution is added slowly. The resultant mixture is stirred for 12 hours. The product is extracted twice with 50 ml portions of dichloro-methane. The combined dichloromethane extract is dried over anhydrous Na2S047 filtered and concentrated on a rotary evaporator to yield 2.0 g of brown oil. This oil is purified by silica gel column chromatography to give o.78 g of 3-dodecylthio-2-propynenitrile (yield 53 percent) which is 80 percent pure by GC/MS.
Antimicrobial Activit~
The compounds of the present invention are useful because of their antimicrobial activity and can be used as antibacterial and/or antifungal agents.
Their effectiveness varies with the concentration of the compound used and the particular organisms to be controlled. While not all compounds are effective at the same concentrations, all the compounds of the present invention are useful as antimicrobial agents in the methods described herein.

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WO91/11503 2~7~4~ RcT/~r ~f~2 -TABLE I
Identification of Compounds used in Antimicrobial Activity Tests Compound Chemical Identity A 3-methylthio-2-propynenitrile B 3-ethylthio-2-propynenitrile C 3-cyclopentylthio-2-propynenitrile D 3-(4-chlorophenylthio)-2~propynenitrile E 3-decylthio-2-propynenitrile F 3-dodecylthio-2-propynenitrile The antimicrobial activity of the compounds of the present invention is demonstrated by the following techniques.

The minimum inhibitory concentration (MIC) for the compounds listed in Table I 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 0.04M solution of N-[tris-(hydroxymethyl)methyl]-glycine buffered deionized water with concentrated sodium hydroxide to a pH of 8.5O
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 ;.: , . , : . . .
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_19_ 25 x 200 mm test tubes, capped and autoclaved for 15 minutes at 115C. The test tubes containing the agar are cooled in a water bath until the temperature of the a;gar is 48C. 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 30C 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 three wells (i.e., 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.

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The inoculated petri plates are incubated at 3t)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 30C.
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 (TRITON~ X-100, a trademark of Rohm ~ Haas Company) 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 3OC and read after 48 hours for yeast and 72 hours for fungi.
Table II lists the bacteria, yeast and fungi used in the MIC test described above along with their respective American Type Culture Collection (ATCC) identification numbers.

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TABLE II
Organisms used in the Minimum InhibitorY Concentration Test Or~anism ATCC No.
Bacteria Bacillus subtilis (Bs) 8473 Enterobacter aerogenes (Ea) 13048 Escherichia coli (Ec) 11229 Klebsiella pneumoniae (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 In Tables III and IV, the MIC values of the compounds de~cribed in Table I as compared to the MIC of a standard commercial preservative (DOWICIL~ 75, a trademark of The Dow Chemical Company, with 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane :.:- , ' ~- : ;: . - : .
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chloride as the active agent) are set forth for the bacteria organisms and yeast/fungi organisms which are listed in Table II.

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,~ _ _ _ _ _ _ WO 91/11503 ~ ~ 7 3 ~ ~8 p~ J~g~ u TABLE IV
Minimum Inhibitory Concentrations for Test Compounds in Yeast/Fun~i sPecies (in ppm) ORGANISMS
COMPOUND
An Ca Pc Sc Tv Ap Fo ~ -DOWICIL0 75 >500 >500 >500 500 >500 >500 >500 ~ 10 A 2.5 0.50 0.25 0.500.252.5 0.50 ; B 2.5 <1.0 <1.0 <1.0 10<1.0 <1.0 C 2.5 2.5 <1.0 2.5 2.52.5 <1.0 D <1.0 <1.0 <1.0 <1.02.5<1.0 <1.0 E >500 >500 500 25 500>500 500 F >500 >500 500 >500 >500 >500 >500 .
Additionally, the ability of the compounds of Table I to serve as preservatives in a variety of formulated industrial, household, and commercial products is tested using a Multiple Challenge Test Protocol. In this test, the formulations include a styrene-butadiene latex, a tape joint, a hand lotion, 25 and a shampoo. The styrene-butadiene latex test formulation used is Latex DL 238A, a~ailable from The Dow Chemical Company. The compositions of the tape - joint, hand lotion and shampoo test formulations can be found in Tables V-VII.

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W091/11503 2 0 7 3 ~ ~ ~ PC~J1JS9~00262 TABLE V t Tape Joint Test Formulation Raw MaterialPweerlc~ehntt Calcium Carbonate 60.00 Mica 4.00 Attapulgite Clay 2.00 Hydroxypropyl 0.40 Methylcellulose Polyvinyl acetate Latex 3.00 Ethylene Glycol 0,34 Water 30.24 Defoamer 0.02 TABLE VI
Hand Lotion Test Formulation Raw Material Percent Deionized Water 78.60 Carboxymethylcellulose 0.15 Propylene Glycol 5.00 Stearic Acid 3.00 Stearyl Alcohol 1.00 Cetyl Alcohol 0.50 Glyceryl Monosterate 4.00 Mineral Oil . 5.00 Silicone 0.50 Deionized Water 2.00 Triethanolamine 0.25 i. . , . . . ~:

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WO91tll503 2~3~,S,8., ,, Pc~ Js~

TABLE VII
Shampoo Test Formulation Raw Material Percent Deionized Water 60.00 Sodium Lauryl Ether Sulfate 30.00 Hydrolyzed Keratin Protein 1.00 Hydrolyzed Animal Protein4.00 Cocamide Diethanolamine 2.00 Cocamidopropyl Betaine 1.00 Sodium Chloride 0.50 Citric Acid qs The formulations are separated into 50 g aliquots and placed in sterile bottles. An appropriate amount of a fresh one percent stock solution of the test compound in acetone-water is added to aohieve the desired final concentrations. A small portion of each of the test formulations is streaked onto Tryptic Soy Agar (TSA) petri plates using sterile cotton swabs to ensure that the formulations are sterile. If the formulation is sterile, then it is inoculated with 0.1 ml of a mixture of equal aliquots of the 24 hour cultures of each of the bacterial organisms listed in Table II. The test formulations are then incubated at 30C. After 24 hours, each sample is streaked onto a TSA petri plate using a sterile swab. All plates are then incubated at 30C for 48 hours and then rated for ; microbial growth using the rating system listed in Table VIII. Samples with a rating of 3 or less are reinoculated as de~cribed in the procedure for the first inoculation. Sample~ with a rating of 4 or greater are .
:: - . . ., ....................... --WO 91 /t 1503 2 o 7 3 4 5 8 PC~tlJSg~tO~?~
, not reinoculated but after another 24 hours are restreaked on TSA agar.
TABLE VIII
Microbial Growth Ratin~ Svstem Ratin~ No. of Colonies 9 Too Many To Count Solid Mass The normal interpretation of the results is that a concentration of the antimicrobial being tested is considered to be effective if no ratings greater than a 3 are observed throughout the 10 challenges. The Minimum Effective Concentration (MEC), which is used to compare the activity of the antimicrobial additives in the end-use formulation~, is the minimum level that is effective under the conditions of the Multiple Challenge Test.
3 Under the conditions of the Multiple Challenge test, good protection is obtained in styrene-butadiene latexes used for paper coatings, inks, adhesives, soaps, cutting oils, and textiles when the compounds of the invention are added at 0.001 to 0.025 percent by weight of the formulation. An example of the effectiveness of : , .

.:
, W091/11503 ~ 5 ~ . PC~'JFJS5~ 2 ~ 1.

r the compounds of the invention as compared to a commercial standard in a particular styrene-butadiene latex is listed in Table IX.
TABLE IX
5 MEC of Test Compounds in Styrene-Butadiene Latex Formulation (in pDm) Compound MEC
DOWICIL0 75 250 ppm A 10 ppm B 25 ppm C 100 ppm D 100 ppm In the preservation of latex-based paints and tape joints against the destruction caused by growth of bacteria and fungi, the compounds of the invention are added at con¢entrations of at least 0.002 percent by weight. Thl~ effectiveness can be compared to that of the commercial standard (DOWICIL0 75) in a tape joint compound, under the conditions of the Multiple Challenge Te~t, in the results in Table X.

.,.

.. .. . . . ..

W091/11503 ~ 4 ~ ~ PCT/~JS9~/QO?62 TABLE X
MEC of Test Compounds in Tape Joint Formulation (in Ppm) Compound MEC
DOWICIL0 75 400 ppm A 10 ppm B 25 ppm C 100 ppm D 100 ppm In the preservation of personal care products like hand creams, lotions, shampoos, and hand soaps 9 good protection is obtained at concentrations from 0.001 to 0.03 percent by weight of the formulation. The results in Tables XI and XII confirm the effectiveness of the compounds as compared to the commercial standard (DOWICIL~ 75) under the conditions of the Multiple Challenge Test.
TABLE XI ', MEC of Test Compounds in Hand 25Lotion Formulation -` (in PPm) Com~ound MEC -~:
:~ DOWICIL~ 75 250 ppm 3 A 10 ppm ; B 25 ppm C 250 ppm D 250 ppm '.

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WO91/11503 - ' ~ RCT/US91/00262 2073~3~8 ~ ~

TABLE XII
MEC of Test Compounds in Shampoo Formulation (in ppm) Compound MEC
DOWICIL~ 75 750 ppm A 10 ppm B 25 ppm C - 100 ppm D 100 ppm Mixture of a Substituted 2-Chloro-3-Thio--2-Propenenitrile and a Like-Substituted 3-Thio-2-Prop~nenitrile An appropriately substituted 2-chloro-3-thio--2-propenenitrile will generally exist as a reaction product with both an E isomer and a Z isomer, as shown below, wherein R ls as defined hereinabove.

\C C / \C C /
H / \ Cl H / C=N
E I~omer Z I-~omer The ratio of E isomer to Z isomer of an appropriately substituted 2-chloro-3-thio-2-propenenitrile reaction product is largely dependent on the reaction process and conditions employed to produce the appropriately substituted 2-chloro-3-thio-2-propenenitrile. As such, an appropriately substituted 2-chloro-3-thio--2-propenenitrile reaction product will generally exist with 50 to 10 weight percent E isomer and 50 to 90 . ~ ~ - ' ,........................................ . -, - , , wo9~ so3 PCTIUS~ 0~62 2073.~

weight percent Z isomer, based on a total weight of the appropriately substituted 2-chloro-3-thio- '!
-2-propenenitrile. A typical appropriately substituted 2 chloro-3-thio-2-propenenitrile reaction product will generally exist with 25 weight percent E isomer and 75 weight percent Z isomer.
It has also been discovered that the Z isomer of an appropriately substituted 2-chloro-3-thio- ~ --2-propenenitrile precursor may degrade in situ to a like-substituted 3-thio-2-propynenitrile. The half-life for the degradation of the Z isomer of 2-chloro--3-methylthio-2-propenenitrile to 3-methylthio--2-propynenitrile has been found, for example, to be approximately two days at pH 9 and approximately two months at pH 7.
The pure E and the pure Z isomers of 2-chloro-3-methylthio-2-propenenitrile are isolated by flash column chromatography and tested for antimicrobial activity. Table XIII identifies the compounds or mixtures of compounds used in the following antimicrobial activity tests. The ratio mixtures for Compounds G, J, K, and L are ba~ed upon weight percent.

.

.. , .. - . .. .

W091/11503 P~T/~JS91/00262 ,, 2~734~

TABLE XIII
Identification of Compounds used in Antimicrobial Activit~ Tests Compound Chemical Identit~

A 3-(methylthio)-2-propynenitrile G 2-chloro-3-(methylthio)-2-propenenitrile (approximately 25:75 ratio mixture of E isomer to Z isomer) H E isomer of 2-chloro-3-(methylthio)-2-propenenitrile I Z isomer of 2-chloro-3-(methylthio)-2-propenenitrile J 90:10 ratlo mlxture of Compound G to A
K 80:20 ratio mixture of Compound G to A
L 50:50 ratio mixture of Compound G to A

The following MIC and MEC antimicrobial tests, whose results are shown in Tables XIII-XVII, are performed using the same methods as described hereinabove.
As can be seen in Table XIV, the Z isomer of ; 2-chloro-3-methylthio-2-propenenitriIe (Compound I) has antimicrobial activity which is essentially equivalent to 3-methylthio-2-propynenitrile (Compound A) and 25-50 times greater than the E iqomer of 2-chloro-3 -3-methylthio-2-propenenitrile (Compound H) at a pH of 8.2, due to the partial conversion of the Z isomer to 3-methylthio-2-propynenitrile.

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l ~D 0 ~O 0 ~.D 0~D 0 ~,o 0 t~O t;l~ ~,0 0 C 2 ~ ~1 l L~. 0~ CL ~ ~ -O _ _ _ _ _ _ _ t,_ '~

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wo9l/11503 ~ PCT/US91/00262 2 ~ 8 ' ' TABLE XV
Minimum Inhibitory Concentrations for Test Compounds in YeastiFun~i sPecies (in pPm) ORGANISMS
COMPOUND
An Ca Pc Sc Tv Ap Fo ----A 2.50.500.25 0.50 0.25 2.5 0.50 J 5 10 2.5 5 10 S 5 K 1 2.5 1 1 5 5 L 1 2.5 1 2.5 2.5 It has also been discovered that the compounds of the present invention may degrade at a pH of 7 or above over an extended time period to compounds which are less antimicrobially active than the original compounds of the present invention. For example, 3-methylthio-2-propynenitrile is observed to decompose.
in a basic solution to several compounds, one of which is 3,3-bis(methylthio)propenenitrile, repre~ented below, which is virtually inactive as an antimicrobial.

CH3-S C=N
C=C /

.

Antimicrobial compositions containing both an appropriately substituted 2-chloro-3-thio-,' . ' , , ' : ' -.. ~ . . . -:, ... . . . .

, ~ : . -W091/l1503 ~ T~J~fs~o~
2~734~8 '' -2-propenenitrile precursor and a like-substltuted 3-thio-2-propynenitrile are formulated. Such antimicrobial compositions generally exhibit an increased potency of antimicrobial activity as compared to the appropriately substituted 2-chloro-3-thio--2-propenenitrile precur~or alone and also result in a more stable level of the like-substituted 3-thio-2-propynenitrile than the like-substituted 3-thio-2-propynenitrile alone, thus generally resultirlg in greater antimicrobial activity over an extended time period.
Such an antimicrobial composition containing both an appropriately substituted 2-chloro-3-thio--2-propenenitrile precursor and a like-substituted 3-thio-2-propynenitrile will contain between 50-95 weight percent, preferably between 80-90 weight percent, of the appropriately substituted 2-chloro--3-thio-2-propenenitrile precursor and between 50-5 weight percent, preferably between 20-10 weight percent, of the like-substituted 3-thio--2-propynenitrile, based on a total weight of the appropriately substituted 2-chloro-3-thio--2-propenenitrile precursor and the like-substituted 3-thio-2-propynenitrile.
A mixture of an appropriately substituted 2-chloro-3-thio-2-propenenitrile precursor and the like--substituted 3-thio-2-propynenitrile, or a mixture of 3 two or more such appropriately substituted 2-chloro--3-thio-2-propenenitriles precursors and the like-substituted 3-thio-2-propynenitriles, must be present in an antimicrobial composition in an amount needed to be antimicrobially effective so as to exhibit inhibition of selected organisms. As such, both the .

... . . ............................................. .
' WO91/11503 2 B 7 3 ~ ~ ~ ` PCTflJ~9~ ~5~

-36~

appropriately substituted 2-chloro-3-thio--2-propenenitrile precursor and the like-substituted 3-thio-2-propynenitrile will be present in the mixture ill amounts sufficient to make the mixture antimicrobially effective. In e~sence then, both the appropriately substituted 2-chloro-3-thio--2-propenenitrile precursor and the like-substituted 3-thio-2-propynenitrile will be present in the mixture in antimicrobially effective amounts. However, becau~e of the benefits of using both the appropriately substituted 2-chloro-3-thio-2-propenenitrile precursor and the like-substituted 3-thio-2-propynenitrile together, the amounts of each of the appropriately substituted 2-chloro-3-thio-2-propenenitrile precursor and the like-substituted 3-thio-2-propynenitrile used in the mixture will generally be less than the amount of the appropriately substituted 2-chloro-3-thio--2-propenenitrile precursor and the like-substituted 3-thio-2-propynenitrile needed when used separately to achieve the same level of both short- and long-term antimicrobial activity as the mixture.
Typically, the amount of such a mixture to be used varies from 1 part per million (ppm) to 5000 ppm by weight. Such amounts vary depending upon the particular mixture tested and organism treated. Also, the exact concentration of the mixtures to be added in the treatment of industrial and consumer formulations may vary within a product type depending upon the components of the formulation. In such formulatio~s, the mixtures of this invention may be added as a liquid concentrate or diluted with additional liquid to produce the ultimate treating composition, wherein the liquid could .
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WO91/11503 J ~ PCT

be water or an organic solvent like glycols, alcohols, or acetone.
The increased rate of antimicrobiaL activity exhibited by mixtures of 2-chloro-3-methylthio--2-propenenitrile and 3-methylthio-2-propynenitrile (Compounds J, K and L) as compared to 2-chloro--3-methylthio-2-propenenitrile alone (Compound G) is demonstrated by the MIC test data shown in Table XIVo As shown in Table XVI, in a tape joint formulation with a pH between 5 and 7, after 3 months storage at room temperature, the mixtures of 2-chloro-3-methylthio-2-propenenitrile and 3-methylthio-2-propynenitrile (Compounds J and K) protect the tested formulation at a lower level than - either the 2-chloro-3-methylthio-2-propenenitrile (Compound G) or the 3-methylthio-2-propynenitrile (Compound A) alone. The composition of the tape joint formulation can be found in Table V.
TABLE XVI
MEC of Test Compounds in Tape Joint Formulation (in DDm) Compound MEC
A ~50 ppm G ~50 ppm J 25-50 ppm K 25-50 ppm .
~ A~ shown in Table XVII, in a paint formulation with a pH between 7 and 9, after 1 month storage at room temperature, the mixture~ of 2-chloro-3-methylthio-:
. .

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WO91/11503 - ~ PCT/US91/00262 2Q7~5~ ~
-~8- ., -2-propenenitrile and 3-methylthio-2-propynenitrile (Compounds J and K) protect the tested formulation at a lower level than the 3-methylthio-2-propynenitrile alone (Compound A). The composition of the paint formulation can be found in Table XVIII.

TABLE XVII
MEC of Test Compounds in a Paint Formulation (in ppm) Compound MEC
A >50 ppm J 25 ppm K 25 ppm ;

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Paint Test Formulation Raw Material Wei~ht Hydroxyethyl cellulose O.3 Ethylene Glycol 2.1 Water 21.7 Acrylic Polymer Dispersing Agent O.9 Potassium Tripolyphosphate 0.1 Octylphenoxypolyethoxyethyl 0.2 Benzyl Ether Nonionic Surfactant Silicone Defoamer 0.3 Propylene Glycol 2.9 Titanium Dioxide 21.1 :.
Talc 17.2 Acrylic Latex 32.1 2,2,4-Trimethyl-1,3-Pentanediol 0.9 Monoisobutyrate Ammonium Hydroxide O.2 ~ -:

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Claims (11)

1. A compound corresponding to the formula:
R-S-C?C-C?N
wherein R is an alkyl, cyclic alkyl, aryl, or heterocyclo group.

2. The compound of Claim 1 wherein R is a straight chain alkyl of 1 to 18 carbon atoms, a branched chain alkyl of 3 to 18 carbon atoms, or a cyclic alkyl of 3 to 8 carbon atoms.

3. The compound of Claim 1 wherein R is methyl, ethyl, propyl, butyl, decyl, dodecyl, isopropyl, tertiary butyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, chlorophenyl, thiazolyl, triazolyl, imidazolyl, or pyrimidyl.

4. An antimicrobial composition comprising a liquid diluent and a compound corresponding to the formula:
R-S-C?C-C?N
wherein R is an alkyl, cyclic alkyl, aryl, or heterocyclo group.

5. The composition of Claim 4 wherein R is a straight chain alkyl of 1 to 18 carbon atoms, a branched chain alkyl of 3 to 18 carbon atoms, or a cyclic alkyl of 3 to 8 carbon atoms.

6. The composition of Claim 4 wherein R is methyl, ethyl, propyl, butyl, decyl, dodecyl, isopropyl, tertiary butyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, chlorophenyl, thiazolyl, triazolyl, imidazolyl, or pyrimidyl.

7. The composition of Claim 4 further comprising the appropriately substituted 2-chloro--3-thio-2-propenenitrile precursor of the compound.

8. The composition of Claim 7 wherein the compound is 3-methylthio-2-propynenitrile and the appropriately substituted 2-chloro-3-thio--2-propenenitrile precursor is 2-chloro-3-methylthio--2-propenenitrile.

9. The composition of Claim 8 wherein the 3-methylthio-2-propynenitrile is 50-5 weight percent and the 2-chloro-3-methylthio-2-propenenitrile is 50-95 weight percent based on a total weight of the 2-chloro-3-methylthio-2-propenenitrile and the 3-methylthio-2-propynenitrile.

10. A process for the preparation of the compound of Claim 1 comprising:
reacting substituted 2-chloro-3-thio--2-propenenitrile precursor with a Lewis base in the presence of an inert, water-miscible solvent.

11. A process of using a compound corresponding to the formula:

R-S-C=C-C=N
wherein R is an alkyl, cyclic alkyl, aryl, or heterocyclo group as an antimicrobial agent.