Note: Descriptions are shown in the official language in which they were submitted.
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Wo91/l1503 PCT/US9~,~Q0262
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COMPOSITION AND USE OF SUBSTITUTED
3-THIO-2-PROPYNENITRILES
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
-2-
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:
~ . - - , , . , , - , .
. , ~ , . . . .
: .
,
,:' ' ' ' ' :
WO91/11503 ~ ~ ~ ; 2 0 7 3 4 5 8 PC~
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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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WO 91/11503 : - ' 2 ~ ~ 3 ~ 5 ~ Pcr/~Js~c~
-4-
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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WO91/11503 ~ PCT/US91/0026~ l
~3 !
-6-
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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WO 91/11503 -~ ~ R~/US~fD~
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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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.
WO91/11503 2~73~5~ RcT/nJ~ 262
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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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WO 91/1 1503 , ~ PCrJ~]S~
--1 0--
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
CH3-S-C--C-CN
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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- ' . : - ' . : , '
WO 9 1 / 1 1 503 2 ~ ~ 3 ~ 5 8: - pC~J~? 1` ~ ]~
.,,, --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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WO91/11503 2 Q7 3 ~ RCTJ~S91/~ 2.
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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.
.
t~ 2 ~ 7 3~ ~ 8 p~ Jssnf~ fi2
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-13-
Example 3 - Preparation of 3-t-ButYlthio-
-2-Propynenitrile
H3C
H3C ~ - S-C~C-CN
H3C
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
-14-
Example 4 - Preparation of 3-Cyclopentylthio-
-2-Propynenitrile
S-C3C-CN
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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WO 9 1 / 1 1 503 , . : PCr/l~
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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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WO 91/11503 2 ~ 7 3 ~ 5 8 pcrJr)~2!~ r~
-16-
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.
,~ ' :: . ,~
WOs1/11503 2~734~8 PCT~J~
-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
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-18-
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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: - . - . :
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WO91/11503 2~34~`8 ` - i p~ 6~
_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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WO 91 /1 1503 ~ ~ 7 3 ~ 5 g ~ s ~ ) ? ~ I ? ~
-20-
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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. .
:- - :
WC~ 91/1 1503 2 3-7 3 4 S 8 P(~,'f l~i~' J'~(~2a;~
--2 1--
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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W091/11503 ~i ".~.~ PC~.~Y)~ 2
- 2~ `8 `" ~ ~
-22- 1
I
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 9~ 503 2 ff!~ 7! 3~ 8; PCr/US9~ 0262
- 2 3 -
o o u~ o o O o o o ~ In u~ O m
~5o Lt~ N N N U~ N
v~ - N N U~ ~ l~ V V V A
~ ~1 ~ _ ~ ~ n ~ ~ f~
~ o o ~ U~ Ul N O O O
O " al O O ~ U~ ~n ,~ O O ~ O O
o c ~,o o In , o o o o o O O O O
H O ~ H I f ~ V N V u~ V V V U~ Ln m
H .~ _1
H ~J O ~ ~ O 0 ~1 0 0 0 0 ~ O ~ o o o o
CC fl~ fd
l n u~ o O _ v ~ u~
C
S dl O U~ o o -- ~
iH
e O O ,~ O U~ ~ O O O O O U~ O O
~ ,7~ ,-n m N N O U~ O f~U O O
O m N N 11~ -- N V -- -- --
~ ~ Ir~
'a t-- 0 N a:~ N 0 N 0 ~J f~) N 0 f~U f O N
C f0 D 0 ~D 0 ~.0 f ~ ~ 0 ~O f ~ ~ f ~ `D 0
ff f,) ~ ~ f f~ ~
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WO 91/11503 ~ ~ 7 3 ~ ~8 p~ J~g~ u
-24-
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
-25-
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. . , . . . ~:
:, ,, . ,' :: .'.` :, . : .. ..
WO91tll503 2~3~,S,8., ,, Pc~ Js~
-26-
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~?~
,
-27-
not reinoculated but after another 24 hours are
restreaked on TSA agar.
TABLE VIII
Microbial Growth Ratin~ Svstem
Ratin~ No. of Colonies
2 1-4
3 5-10
4 11-25
26-50
6 51-100
7 101-200
15 8 201-300
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
: ,
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.:
,
W091/11503 ~ 5 ~ . PC~'JFJS5~ 2
~ 1.
-28-
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.
.,.
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W091/11503 ~ 4 ~ ~ PCT/~JS9~/QO?62
-29-
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
'.
:..... :. ~ .. . .. ,,, , - . ~ - - ; ~ --
-.,: ~ , - : ~ - ... :- - ; :
.. . . .
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
. ~ ~ - '
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,
- , ,
wo9~ so3 PCTIUS~ 0~62
2073.~
-31-
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.
.
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W091/11503 P~T/~JS91/00262 ,,
2~734~
-32-
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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.
: . .. ... .. . ..
WO 91/1 1503 2 ~ 7 ~ ~ 5 ~ t ' ' . PC~ t!5~0262 11
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u~ ta LO ta ta U~ ~ ta ~ ta
t I U~ ta L~ O ta ta 1~ ta ~
C
e t~ ta L~ ta ta Lr~ ta L~ ~ O In
c~ :~ ~
E-. E ~ Ln ta ta ta ta m Lr~ ta "~
t;~
C ~ I ~ ta u~ " ~ ta ~ ta U~ ta Ln
m c V~ O ~ ~ _ _ Ln ta U~ O ~
~ c t~
L ~ t I U~ ~r ~
s l "'I u~ o~ o o o o N h~ N ~ ~ I
L 01 m m m _ o o o ta U~ _ _ -- u~ --
t,~l 0 t~ 0 t~'~J 0 t~l 0 t~l 0 ~I 0 ~
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 _ _ _ _ _ _ _
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wo9l/11503 ~ PCT/US91/00262
2 ~ 8 ' '
-34-
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
10 G 50 50 50 100 100 50 50
H 250250 100 500 250 250 100
I 50 100 50 100 50 50 100
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 /
CH3 S H
.
Antimicrobial compositions containing both an
appropriately substituted 2-chloro-3-thio-
,' . ' , , ' : ' -
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:, ... . . . .
, ~ : . -
W091/l1503 ~ T~J~fs~o~
2~734~8 ''
-35-
-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
.
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'
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
37-
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-
:
. .
,
:- . - . . ,
.~ . . . . , . ~ . , .
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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WO91/11503 2073458 ~ PCT/.~1S91/Q~262 ~,
-39-
TABLE XVIII
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
~ -:
,. ~:.. . , . . ... .. , : ~ . : - .
