Antibacterial product

A number of water-soluble, one-to-one quaternary ammonium adducts of unsaturated organic halides and dihalides with hexamethylenetetramine are known as being useful as antimicrobial agents. It has now been discovered that a two-to-one quaternary ammonium adduct can be prepared from hexamethylenetetramine and 3,4-dichlorobutene-1; this two-to-one adduct is a more active, less toxic antimicrobial agent than the known one-to-one quaternary ammonium adducts.

BACKGROUND OF THE INVENTION 
During 1915 and 1917 a number of papers were published by Dr. Walter Jacobs 
and coworkers at the Rockefeller Institute for Medical Research describing 
the general bactericidal character of quaternary ammonium salts of 
hexamethylenetetramine. The bacteridical activity of these compounds was 
primarily attributed to the presence of the hexamethylenetetramine 
nucleus; however, the degree or extent of this activity was said to be 
determined by the nature of the molecular groups adducted with the 
hexamethylenetetramine. The principal mode of action of these quaternary 
ammonium salts of hexamethylenetetramine was said to be the controlled, 
regulated decomposition of the hexamethylenetetramine nucleus to liberate 
formaldehyde. The following groups of papers by Jacobs and coworkers are 
of interest in this regard: Proc. Nat. Acad. Sci. U.S.A., 1, 226 (1915); 
J. Biol. Chem., 20, 659 (1915); J. Biol. Chem., 21, 465 (1915); J. Exp. 
Med., 24, 563 (1916); and J. Exp. Med., 25, 363 (1917). These Jacobs 
papers, however, do not describe any quaternary ammonium adducts of 
hexamethylenetetramine with unsaturated acyclic dihalides. 
In U.S. Pat. No. 3,228,829 Wolf and a coworker at the Dow Chemical Company 
have more recently described as antimicrobial agents a group of one-to-one 
quaternary ammonium adducts of hexamethylenetetramine with unsaturated 
halides and dihalides. Among the useful unsaturated halides and dihalides 
described are the dihaloalkenes and haloalkynes, specifically 
1,3-dichloropropene. 
In French Pat. No. 1,363,240 Frank and coworkers at Farbenfabriken Bayer 
even more recently described as antibacterial agents a group of one-to-one 
quaternary ammonium adducts of hexamethylenetetramine with 
.alpha.,.omega.-dihalogenated hydrocarbons. Among the group of useful 
.alpha.,.omega.-dihalogenated hydrocarbons described is 
1,4-dichlorobutene-2. 
We have now discovered that a two-to-one quaternary ammonium adduct can 
unexpectedly be prepared by reacting hexamethylenetetramine with 
3,4-dichlorobutene-1. Moreover, this two-to-one quaternary ammonium adduct 
is more active and less toxic that the one-to-one adducts described by 
both Wolf and Frank and consequently is significantly more advantageous as 
an antimicrobial agent, particularly from a safety and environmental 
standpoint. 
BASIC AMETERS OF THE INVENTION 
A. Preparation of the Product 
When hexamethylenetetramine and the dihalides of Wolf and Frank react, only 
one of the halogens is displaced by hexamethylenetetramine to form a 
quaternary ammonium salt. The second halogen, however, is unreactive and, 
therefore, only a one-to-one quaternary ammonium adduct is formed. On the 
contrary, when hexamethylenetetramine and 3,4-dichlorobutene-1 react, both 
chlorines are displaced by the hexamethylenetetramine so that a two-to-one 
quaternary ammonium adduct is formed. 
Contrary to the reaction of the Wolf and Frank dihalides, it is believed 
that the reaction of this invention between hexamethylenetetramine and 
3,4-dichlorobutene-1 proceeds by a rearrangement mechanism which results 
in the presence during the reaction of two reactive sites on the dihalide 
and thus allows for the preparation of the two-to-one rather than the 
one-to-one quaternary ammonium adduct. The reaction is believed to occur 
in the following fashion, with the rearrangement being accomplished by the 
allylic displacement of the 3-chlorine and concomitant shift of the double 
bond: 
##STR1## 
Regardless of the actual mechanism of the reaction, the analytical data 
available on the product clearly indicates that the two-to-one quaternary 
ammonium adduct is formed. Particularly, the analyses for percent ionic 
chlorine and percent nitrogen in the final product indicate that the 
two-to-one quaternary ammonium adduct is obtained according to this 
invention. Moreover, nuclear magnetic resonance and infrared data indicate 
the product is symmetrical about the double bond and free of vinyl groups 
and these data are thus confirmatory of the formation of the above 
depicted symmetrical two-to-one adduct. 
The reaction of hexamethylenetetramine and 3,4-dichlorobutene-1 to form the 
two-to-one quaternary ammonium adduct of this invention should 
advantageously be carried out in an inert organic solvent in which the 
reactants are substantially soluble and the product is substantially 
insoluble. Suitable solvents are hydrocarbon and chlorinated hydrocarbon 
solvents such as chloroform and perchloroethylene. 
The reaction should be conducted between 0.degree. C and a maximum upper 
limit of about 130.degree. C., the decomposition point of the two-to-one 
quaternary ammonium adduct. There are no necessary pressure conditions for 
the reaction, and the reaction can thus be carried out at sub, super or 
normal atmospheric pressure. However, use of super atmospheric pressure 
reduces substantially the reaction time. 
Consequently, the only two requisite reaction parameters are that (1) the 
reaction be carried out in an inert organic solvent in which the product 
is substantially insoluble and (2) the reaction temperature be maintained 
at less than about 130.degree. C. to avoid decomposition of the product. 
B. Structural Characteristics of the Products 
In view of the disclosure of the Wolf and Frank patents, it is unexpected 
and unpredictable that a two-to-one quaternary ammonium adduct is formed 
by the reaction of hexamethylenetetramine with 3,4-dichlorobutene-1. A 
number of analyses have been conducted upon the product obtained according 
to this invention which confirm that high yields of the two-to-one 
quaternary ammonium adduct are obtained by the process of this invention. 
Table 1 on page 5 represents a comparison of analytical data relating to 
the organic-ionic chlorine content of products obtained according to this 
invention and products obtained by varying the conditions of example 1 of 
the Frank patent. The data in Table 1 confirm that the product of this 
invention is a two-to-one rather than one-to-one quaternary ammonium 
adduct. 
TABLE I 
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Organic 
Organic Ionic Ionic 
Chlorine 
Chlorine Chlorine 
Chlorine 
Calculated 
Calculated Calculated 
Calculated 
Product and Total Total 
On Total 
On Total 
Organic 
On Total 
On Total 
Ionic 
Preparation Chlorine 
Chlorine 
Chlorine 
Chlorine 
Chlorine 
Chlorine 
Chlorine 
Chlorine 
Technique Calculated 
Obtained 
Calculated 
Obtained 
Obtained 
Calculated 
Obtained 
Obtained 
__________________________________________________________________________ 
Repetition of Example 
1 of Frank patent 
26.6% 24.7% 
13.3% 12.4% 10.8% 13.3% 12.4% 13.9% 
Repetition of Example 
1 of Frank patent 
with large excess of 
1,4 dichlorobutene-2 
26.6% 25.9% 
13.3% 13.0% 11.8% 13.3% 13.0% 14.1% 
Repetition of Example 
1 of Frank patent 
using extended reflux 
and large excess of 1,4- 
dichlorobutene-2 
26.6% 25.7% 
13.3% 12.9% 11.7% 13.3% 12.9% 14.0% 
Procedure according to this 
invention using 3,4 dichloro- 
butene-1 17.5% 17.7% 
0 0 0.4% 17.5% 17.7% 17.3% 
Procedure according to this 
invention using 3,4 dichloro- 
butene 1 17.5% 17.1% 
0 0 2.1% 17.5% 17.1% 15.0% 
Procedure according to this 
invention using 3,4 dichloro- 
butene-1 17.5% 17.1% 
0 0 1.0% 17.5% 17.1% 16.1% 
Procedure according to 
this invention using 
3,4 dichlorobutene-1 
17.5% 16.1% 
0 0 0.2% 17.5% 16.1% 15.9% 
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Additionally, determinations of the percent nitrogen in the product of this 
invention confirms that the two-to-one quaternary ammonium adduct is 
formed. Table II is a comparison of the percentages of nitrogen calculated 
and obtained for the product of this invention and the one-to-one 
quaternary ammonium adduct formed according to example 1 of the Frank 
patent. 
TABLE II 
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Nitrogen Nitrogen 
Calculated 
Obtained 
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Repetition of Example 
1 of the Frank patent 
21.1% 22.6% 
Procedure according to 
this invention using 
3,4-dichlorobutene-1 
27.6% 27.8% 
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Nuclear magnetic resonance and infrared analysis confirm that the product 
of this invention is the two-to-one quaternary ammonium adduct, presumably 
prepared according to the rearrangement mechanism outlined hereinabove. 
These data indicate (1) the absence of monosubstituted ethylene or vinyl 
groups and (2) high symmetry about the double bond; both of these 
structural aspects confirm that the final product is completely or almost 
completely made up of the symmetrical two-to-one adduct. However, the 
presence of insignificant amounts of a one-to-one adduct cannot be 
conclusively ruled out by these techniques. 
As a result of the elemental analysis for organic chlorine, ionic chlorine 
and nitrogen and the nuclear magnetic resonance and infrared analysis, it 
has been determined that the product made according to this invention is 
almost completely, if not completely, made up of the symmetrical 
two-to-one quaternary ammonium adduct of 3,4-dichlorobutene-1 with 
hexamethylenetetramine, i.e., 1,1'-(but-2-enylene) 
bis(3,5,7-triaza-1-azoniaadamantane chloride). 
C. Antimicrobial Activity of the Product 
The two-to-one quaternary ammonium adduct of hexamethylenetetramine and 
3,4-dichlorobutene-1 is a more active antimicrobial agent than either of 
the one-to-one quaternary ammonium adducts of Wolf and Frank. The higher 
activity is due to the fact that there are two hexamethylenetetramine 
nuclei in the product of this invention, both of which upon decomposition 
liberate formaldehyde. Consequently, the same amount by weight of the 
two-to-one quaternary ammonium adduct of this invention is more active as 
an antimicrobial agent than the same amount by weight of the one-to-one 
adducts of Frank and Wolf. In this sense more active means that assuming 
that (1) the amount of quaternary ammonium adduct used and (2) the rate of 
formaldehyde release are the same, the antimicrobial activity of the 
two-to-one quaternary ammonium adduct of this invention will be more 
persistent as the two-to-one adduct will release formaldehyde for a 
significantly longer period of time. Consequently the same amount of the 
two-to-one quaternary ammonium adduct of this invention is capable of 
controlling larger numbers of microorganisms. 
Additionally, the two-to-one adduct formed from hexamethylenetetramine and 
3,4-dichlorobutene-1 according to this invention is less toxic than either 
the Frank or Wolf one-to-one adducts. For instance, data comparing the 
toxicological character of the two-to-one adduct of this invention to that 
of both the one-to-one adduct of example 1 of Frank and the one-to-one 
adduct prepared from hexamethylenetetramine and 1,3-dichloropropene 
according to Wolf (available commercially as Dowcil 100 and 200) indicate 
the two-to-one adduct to have a significantly superior toxicological 
profile. 
Table III shows these comparative data for both acute dermal and acute oral 
LD.sub.50 tests. Both the acute oral LD.sub.50 and acute dermal LD.sub.50 
tests were run in accordance with standard, accepted protocols. 
TABLE III 
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Acute Acute 
Product Oral LD.sub.50 
Dermal LD.sub.50 
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One-to-one adduct of hexa- 
methylenetetramine and 1,-3- 
dichloropropene 745 mg/kg* 540 mg/kg** 
One-to-one adduct of hexa- 
methylenetetramine and 1,4- 
dichlorobutene-2 1.6 gm/kg 2.0 gm/kg 
Two-to-one adduct of hexa- 
methylenetetramine and 3,4- 
dichlorobutene-1 3.4 gm/kg 8.0 gm/kg 
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*Data available from Dow Chemical on Dowicil 100. 
**Data available from Dow Chemical on Dowicil 200. 
From the data in Table III, it is apparent that the two-to-one adduct of 
this invention is markedly superior from a toxilogical standpoint than the 
prior art one-to-one adducts. 
D. The Antimicrobial Uses of the Product 
The two-to-one quaternary ammonium adduct of this invention is a 
water-soluble, non-metallic, non-phenolic antimicrobial agent effective 
against a broad spectrum of gram positive and gram negative 
microorganisms. The two-to-one quaternary ammonium adduct of this 
invention is particularly useful as an antimicrobial agent in a wide 
variety of water-based systems such as latex paints, resin emulsions, 
joint cement, adhesives, dispersed pigments and dyes, and other similar 
aqueous compositions. 
The two-to-one quaternary ammonium adduct of this invention is useful in 
the preparation and storage of systems susceptible to deterioration by 
microorganisms such as the common forms of bacteria. The exact 
concentration of the adduct during use is dependent upon a number of 
factors including duration of storage, temperature of storage, etc. 
However, concentrations in the range of 0.05% to 0.20% based upon the 
weight of the composition have been found to be generally effective. It 
should be noted, however, that the minimal inhibitory concentrations 
vis-a-vis the common bacteria are much less than this suggested range for 
use under commercial conditions.

SPECIFIC EMBODIMENTS OF THE INVENTION 
The following Examples are non-limitative embodiments of the invention and 
are included merely as specific examplification of the invention. 
EXAMPLE 1 
To a 1 liter flask equipped with stirrer, thermometer and reflux condenser 
is charged: 
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chloroform 600 mls 
hexamethylenetetramine 
70 grams 
3,4-dichlorobutene-1 125 grams 
sodium carbonate 10 grams 
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The reaction mass is refluxed for 30 hours and then cooled to room 
temperature and filtered. The filter cake is washed three times with a 
total of 750 mls of chloroform and then allowed to air dry. The 
theoretical calculated yield is 111 grams while the actual weight obtained 
is 108 grams. 
EXAMPLE 2 
To a 2 liter stirring autoclave the following charge is added: 
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chloroform 1,020 mls 
hexamethylenetetramine 
119 grams 
butylated hydroxy toluene 
0.184 grams 
3,4-dichlorobutene-1 212.5 grams 
sodium carbonate 8.5 grams 
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The reaction is maintained at 80.degree. C. for a period of 18 hours with a 
maximum generated pressure of 12 psi. At the end of the reaction period, 
the mixture is cooled and filtered. The filter cake is then washed with 
350 ml increments of chloroform three times and then placed in a vacuum 
oven at 40.degree. to constant weight. The theoretical calculated yield is 
180.5 grams while the actual yield obtained is 166 grams. 
EXAMPLE 3 
To a 1 liter flask equipped with stirrer, thermometer and reflux condenser 
is charged: 
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perchloroethylene 600 mls 
3,4-dichlorobutene-1 125 grams 
hexamethylenetetramine 
70 grams 
sodium carbonate 10 grams 
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The reaction is heated at 75.degree. C. for 25 hours. Product mix is cooled 
and then filtered. Filter cake is washed 4 times with 250 ml increments of 
perchloroethylene and then air dried. The theoretical calculated yield is 
111 grams and the actual yield obtained is 104 grams. 
EXAMPLE 4 
The product of this invention had demonstrated especially high 
antibacterial performance in the preservation of latex and emulsion based 
paints. The following is a description of a notable paint formulation. 
To a 1 gallon stainless steel beaker, equipped with a sheer type agitator, 
the following ingredients are charged in sequence: 
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water 849 grams 
anionic dispersant 18 grams 
potassium tripolyphosphate 
41/2 grams 
nonionic surfactant 6 grams 
defoamer 6 grams 
ethylene glycol 60 grams 
two-to-one adduct of this 
3.6 grams 
invention 
titanium dioxide 750 grams 
pigment extender 450 grams 
______________________________________ 
In a separate 100 ml beaker, the following premix is prepared: 
______________________________________ 
hydroxyethylcellulose 
9 grams 
coupling solvent 24 grams 
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This premix is then added to the above with agitation such that excessive 
air is not incorporated. To the above prepared combination is added 1,401 
grams of a vinyl acrylic resin emulsion and mixed for a period of 10 
minutes. 
The resulting paint prepared above was examined for antibacterial 
properties by inoculating a portion with approximately 10.sup.8 organisms 
per ml Pseudomonas aeruginosa. Paint samples returned to sterility within 
24 hours of inoculation and maintained the same degree of preservation for 
periods in excess of 9 months, at which time the testing was discontinued. 
The addition of test compound preserved the paint system against attack 
from microbial contamination and thereby maintained the viscosity, 
stability and performance characteristics of the paint. 
EXAMPLE 5 
The following is a formulation for an oil-water hair dressing emulsion: 
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Part A 
Petrolatum 5.5% 
Mineral Oil 35.0% 
Bees Wax 4.5% 
Lanolin Derivative Emulsifier 
4.0% 
Nonionic Emulsifier 2.0% 
Part B 
Water 48.3% 
Part C 
Two-to-one adduct of this 
invention 0.2% 
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Part A is heated to 75.degree. C. and Part B is heated to 70.degree. C. The 
test compound (Part C) is dissolved in Part B and the resulting solution 
added to Part A with good agitation. The mixture was perfumed 45.degree. 
C. and agitated until cold. 
The composition prepared above was tested for microbial stability for a 
period of 6 months and after 6 months at 35.degree. C. Innoculation with 
bacterial cultures at this point showed the material still resistant to 
bacterial contamination.