Note: Descriptions are shown in the official language in which they were submitted.
~07346~
Specification
This invention relates to a new class of micro-
biocidal agents. More particularly, this invention relates
to linear quaternary ammonium polymers in which the quater-
nary ammonium moieties are part of the linear polymeric
chain rather than appendages to, or part of branches on ~-~
the linear chain. Even more particularly, this invention
relates to such linear polymeric quaternary chains in which
the chains terminate in quaternary ammonium moieties, thereby
making further chain propogation impossible. Such polymers ~-
may, therefore, be called "capped" polymers.
The products of this invention can be made by
either a two step reaction or a one step reaction. --
In the first step of the two step reaction a
difunctional tertiary amine is condensed with slightly
more than the molar equivalent of a 1,4-dihalo-2-butene,
after which the unreacted dihalo compound is removed, and
the reaction is completed in a second step by adding a
calculated quantity of monofunctional tertiary amine to
react with the halogen termini of the polymer quaternary.
An excess of the dihalo compound is required in the first
step for the purpose of producing polymer with ha~Qgen
termini.
If the difunctional amine is represented by
N(R') (Rl'~ ZN (R') (R'') and the 1,4-dihalo-2-butene by
X-CH2-CH=CH-CH2-X, where X is a halogen, then if the 1,4-
dihalo-2-butene were not in e~cess, the first step of the
reaction would be represented by the chemical equation:
_
-- .: .: ,
- -
~073~6~
n (R') (R'') N-Z-N (R') (R'') + n X-CH2-CH-CHCH2X
~ r+ + I
t N R' R'' Z N R' R'' - CH2 -CH = CH - CH2 - - 2nX~
_ n
Where X is a halogen such as chlorine or bromine, Z re-
presents either (1) a divalent alihpatic radical of
from 2 to 10 carbon atoms containing from 0 to 2 ethylenic
double bonds and from 0 to 2 hydroxyl substituents, and R'
and R'' may be either (A) the same or different and either
(a) alkyl group of from 1 to 20 carbon atoms having from
0 to 2 hydroxyl substituents, (b) benzyl, (c) benzyl in ~-
which the benzene ring has one alkyl substituent of from
2 to 20 carbon atoms, (d) benzyl in which the benzene
ring has from 1 to 5 methyl substituents; (B) R' and R''
taken together with N form a saturated or unsaturated
heterocyclic ring of from 5 to 7 atoms, or (C) R' and R''
taken together with N may be combined with oxygen to form
a N-morpholino group; or (2) Z represents two divalent
ethylene radicals, in which case RII is absent and R'
represents (a) an aliphatic radical of from 1 to 20 carbon
atoms having from 0 to 2 hydroxyl substituents, (b) ~ ^~
benzyl, (c) benzyl in which the benzene ring has one
alkyl substituent of from 2 to 20 carbon atoms, or (d)
benzyl in which the benzene ring has from 1 to 5 methyl
substituents; or (3) Z represents three divalent ethylene
radicals, in which case RI and RII are both absent.
However, in the presence of a relatively small
excess of 1,4-dihalo-2-butene, the terminal atoms of the
linear polymeric product of the first step are halogen
atoms, the product being:
107346~
G +
X CH2 CH = CH-CH2 ~N R R'' - Z - N R R - CH2CH-CH-CH2J X
where the symbols have the same value or meaning as above. n
Since these linear polymeric chains contain active
halogen termini, the chains can be made to propogate if
more difunctional tertiary amine is added to the reaction ~-
container already holding an excess of 1,4-dihalo-2-butene.
However, if the unreacted dihalobutene is extracted and a
monofunctional tertiary amine is added to the reaction
container after the excess dichloro compound is removed, then
it reacts with the two terminal halogen atoms of the linear
chain to form two terminal quaternary moieties.
This latter reaction constitutes the second step
of the two step process of the present invention, and may
be represented as follows:
X CH2 CH 2 CH CH2 ~ N R' R'' Z N R' R'' -CH2CH~CH-CH~ X
+ 2 N (R''') (RIV) (RV)
{
CH2 - CH = CH CH2 ~ N R''' RIV RV
n
where the symbols have the same value or meaning as above.
The following examples are illustrative of the
two step process.
Example 1
To 42.6 grams of 1~4-bis-(dimethylamino)-2-butene
(0.3 mole) dissolved in 100 ml. of water, there was added
dropwise 50.0 grams of 1,4-dichloro-2-butene (0.4 mole)
with constant stirring. The rate of addition was maintained
lOq346~
at a rate to keep the exothermic reaction at a temperature
of 60C to 70C. ~hen addition was complete, the reaction
mixture was heated on a steam bath at about ~0C to 95C
for more than two hours. Then it was cooled to 20C and
extracted with three 100 ml. portions of ethyl ether to
remove the unreacted 1,4-dichloro-2-butene, and the resi-
due warmed in vacuo to remove ether from the layer con-
taining the polyquatern~ary.
The polyquaternary aqueous layer was analyzed
for total chlorine, and also for ionic chloride. The dif-
ference represented the quantity of terminal chlorine atoms
on the polymeric chains.
The calculations showed that the equivalent of
.03 moles of chlorine terminal atoms remained in the poly-
quaternary chains. Therefore, 4.75 grams of octyl dimethyl
amine (0.03 mole) was added to the aqueous layer, and the
mixture heated on a steam bath for two more hours at about
90C to 95C. The reaction mixture was analyzed for total
polymer quaternary content, and the concentration was
adjusted to 50% active polyquaternary.
The experiment was repeated using petroleum ether
instead of ethyl ether to extract the excess dichloro compound.
The similar results indicated that any inactive organic
solvent may be used to extract the unreacted 1,4-dichloro-
2-butene.
Example 2
In addition to the compound of Example 1, the
following ~Icapped~ polymers were made by substituting other
tertiary amin~es for octyl dimethyl amine, using the procedure
of Example 1. They were as follows:
... . ,. . ., . - .
~0734691
(a) the polymeric quaternary formed by the
reaction of 1,4-bis-(dimethylamino)-2-butene and 1,4-dichloro-
2-butene, capped by decyl dimethyl amine
(b) the polymeric quaternary formed by the reaction
of 1,4-bis-(dimethylamino)-2-butene and 1,4-dichloro-2-butene,
capped by dodecyl dimethyl amine
(c) the polymeric product formed by the reaction
of 1,4-bis~(dimethylamino)-2-butene and 1,4-dichloro-2-
butene, capped by tetradecyl dimethyl amine .
(d) the polymeric product formed by the reaction
of 1,4-bis-(dimethylamino)-2-butene and 1,4-dichloro-2-
butene, capped by hexadecyl dimethyl amine
~xample 3
The following tertiary amines were also used to
cap the polymeric quaternary of 1,4-bis-(dimethylamino) - 2-
butene and 1,4-dichloro-2-butene, using the procedure shown
in example 1:
(a) Propyl dimethyl amine, (b) butyl dimethyl amine,
(c) pentyl dimethyl amine, (d) hexyl dimethyl amine, (e)
dibutyl methyl amine, (f) didecyl methyl amine, (g) di-
octyl methyl amine, (h) quinoline, (i) isoquinoline,
(j) hexamethylene tetramine, and (k) triethanolamine.
Microbiocidal evaluations were made as follows:
Example 4
Each potential microbiocidal capped quaternary
ammoniu~ polymer to be tested was dissolved in distilled
water to the test concentration, and was added aseptically
to previously sterilized cotton-stoppered 125 ml. Erlenmeyer
flasks.
lOq3~64
One set of flasks containing the potential micro-
biocide at concentrations of 25 ppm, 50 ppm, 75 ppm, 100 ppm,
150 ppm, 200 ppm, 250 ppm, and 300 ppm was inoculated by
introducing into each flask 0.5 ml. of a 1/10 nutrient broth
dilution of a 24 hour nutrient broth culture of Aerobacter
aerogenes. Another set of test flasks containing the poten-
tial microbiocide at similar concentrations was inoculated by
introducing into each flask 0.5 ml. of a 1/10 nutrient broth
culture of Pseudomonas aeruginosa.
At intervals of 30, 60 and 180 minutes following
inoculation, a 1 ml. aliquot was withdrawn from each flask
and added to 9 ml. of steril azolection/"Tween 80" ("Tween 80"
is a trade mark) neutralizer from which tenfold serial dilutions
were prepared in the sterile neutralizer solution.
Agarplates were prepared from 1 x 10 2 and 1 x 10 3
dilutions.
Simultaneously with each set of test flasks, a con-
trol of sterile distilled water was similarly inoculated and
aliquots were taken at the same time intervals and plated a
1 x 10 4, 1 x 10 5, and 1 x 10 6 dilutions.
A comparison of the surviving organisms for various
test concentrations of the test material at different time
intervals was made and tabulated.
The test results were as follows:
Table 1
Numher of survivors of A. Aerogenes following expo-
sure to various capped polyquaternaries at different concen-
trations for varying periods of time.
73~6~,
Compound Concentration No. of SurviVing Organisms/ml x 102
in ppm__After 30 Min.60 Min. 180 Min.
Example 1 50 45 10 0
24 4 0
Example 2~a) 50 72 9 0
46 6 0
Example 2(b) 50 24 6 0
12 5 0
Example 2(c~ 50 37 14 0
28 10 0 -
Example 2(d) 50 56 29 0
24 13 0
Untreated Control 45,000 55,000 111,000
Table 2
Number of survivors of P. seruginosa following
exposure to various capped polyquaternaries at different
concentrations for varying periods of time.
Compound Concentration No, of Surviving Organisms/ml x 102
in ppmAfter 30 Min.60 Min. 180 ~5in.
20 Example 150 940 466 0
480 275 0
100 84 26 0
Example 2(a~ 50 750 205 0
230 140 0
100 105 44 0
- Example 2(b) 50 695 137 0
192 59 0
100 82 30 0
Example 2(c) 50 746 150 0
412 81 0
100 65 17 0
1~3~6~
Compound Concentration No. of Surviving Organisms/ml x 10
in ppmAfter 30 Min. 60 Min. 180 min.
Example 2(d) 50 746 176 0
390 89 0
100 74 26 0
Untreated Control 40,000 65,000 90,000
The results of these tests show that the "capped"
polyquaternary ammonium products are very effective micro-
biocides in concentrations at least as low as 50 ppm.
The above-described procedure comprises a two-
step synthesis. In the first step 1,4-dihalo-2-butene is
reacted with slightly less than a molar quantity of a di-
tertiary amine. Under the conditions of this reaction,
polymerization proceeds until the diamine is depleted.
Because of the initial excess of dihalo butene over diamine
in the reaction mixture, the polymeric chains have termini
of halogen atoms, thereby making the ends of the chain re-
active toward the addition of more amine. After the unre-
acted dichlorobutene is removed by extraction at the com-
pletionof the first step, a calculated quantity of mono-
tertiary amine is added to the polymeric residue for thesecond step of the synthesis.
The chemical quaternization which ensues from the
above reaction results in the formation of polymers with
quaternary ammonium termini. Since these quaternized ends
of the chain are incapable of further chain propogation re-
actions with dihalobutene, the polymer is said to be "capped"
in the second step.
--8--
1073~L64
The second step mentioned above merely "caps" the
polymeric products of the first step. Therefore, the chain
lengths and molecular weights of the product are determined
in the first step. Since the polymerization of the first
step proceeds in a sustained fashion until all of the di-
amine is exhausted, the chain lengths are comparatively long
because the number of condensations is comparatively high.
Furthermore, since the propogation of chains is permitted ~-
to proceed uninterruptedly under conditions where each chain
has equal probability to participate in the propogation re- `
action, the product of the first step, and subsequently the
product of the second step, is a mixture of polymeric pro-
ducts whose chain lengths and molecular weights fall within
a comparatively narrow range. -
However, capped polymeric quaternary ammonium
compounds can also be made by a one-step synthesis in which
both ditertiary amine and monotertiary amine are mixed
simultaneously with the 1,4-dihalo-2-butene, there being
about a 1:1 ratio of halogen equivalents to the total number
of tertiary amine equivalents. The molar ratio of diamine
to monoamine in the initial reaction mixture is about 2:1
to about 30:1. The reaction takes placea~ reflux temperature,
which is usually between about 50C - 70C, while the reaction -
time may vary depending on the reactants as well as the
temperature, but is usually about 1 to 10 hours.
In both the one step and two step syntheses the
terminal halogen atoms of a chain may participate in chain
propogation reactions by being displaced by one of the amine
groups of a diamine, thereby making a quaternary nitrogen.
,.
1073~6~
Chain propogation is possible because the second amine of the
diamine is capable of reacting with another dihalo molecule.
However, in the single-step procedure, there is also an al-
ternative route. The terminal halogen of a chain may also react
with a monoamine, instead of a diamine. The reaction with
a monoamine gives rise to a quaternary ammonium terminus
which is incapable of further chain propogation by reaction
with a dihalo molecule. In this manner, any chain may -
become "capped" while other chains are propogating.
Therefore, the product in which both diamine and monoamine --
are used in the initial reaction mixture, is the resultant
of two competing reactions, one a propogation reaction
when the diamine reacts with the terminal halogen and the
other a "capping" reaction, when the monoamine reacts with
the halogen.
In the one step procedure, because of the presence
of monoamine during chain propogation there is always a
possibility that a chain termination reaction will occur
and terminate chain propogation abruptly. Therefore, every
chain cannot grow uninterruptedly. Some chains will con-
tinue to grow while others will terminate, depending on
whether the terminal halogen reacts with a diamine or
monoamine.
Furthermore, some chains are terminated quite
early, leaving only short chains, whereas other chains are
terminated only after having undergone a large number of
propogation reactions, thereby leaving long chains. Termi-
nation occurs in a statistically random fashion. Therefore,
the chain lengths vary from very short to very long, and
--10--
1073~6~
the molecular weights vary from low to high, over a com-
paratively wide range.
The difunctional tertiary amine may be represented ~ -
by the structural formula:
RI\ RII
N - Z - N :-
RI RI
For purposes of clarification, Z represents either (1) ~-
a divalent aliphatic radical of from 2 to 10 carbon atoms
containing from 0 to 2 hydroxyl substituents and from 0 to .~.
2 ethylenic double bonds, and RI and RII may either
be (A) the same or different and may be either (a) an
alkyl group of from 1 to 20 carbo~ atoms having from 0
to 2 hydroxyl substituents, (b) benzyl, (c) benzyl in . .
which the benzene ring has one alkyl substituent of
from 2 to 20 carbon atoms, and (d) benzyl in which the
benzene ring has from 1 to 5 methyI substituents; (B)
RI and RII, taken together with N, form a saturated or :
unsaturated heterocyclic ring of from 5 to 7 atoms; or
(C) RI and RII, taken together with N, may be combined
with an oxygen atom to form a N-morpho~i~ogroup; or
(2) Z represents two divalent ethylene radicals, in
which case RII is absent and RI represents (a) an ali-
phatic radical of from 1 to 20 carbon atoms having from ~ :~
0 to 2 hydroxyl substituents, (b) benzyl, (c) benzyl in
which the bezene moiety has an alkyl substituent of
from 2 to 20 carbon atoms, or (d) benzyl in which the
benzene moiety has from 1 to 5 methyl substituents; or
--11--
, . ... - - ... -, - , , .. . . : ,
~0~3~6~
(3) Z represents three divalent ethylene radicals in
which case RI and RII are both absent.
The monotertiary amine may be represented by
the structural formula:
RIII _ N - RIV -
RV .-
where (1) RIII is an aliphatic radical of from 1 to 20
carbon atoms, having from 0 to 2 hydroxyl substituents,
and RIV and RV may be either (a) the same or different
and represent an aliphatic radical having from 1 to 4
carbon atoms with from 0 to 1 hydroxyl substituents; (b) -~
taken together with N to form a saturated or unsaturated
heterocyclic ring of from 5 to 7 atoms; (c) taken to-
gether with N, and combined with an oxygen atom to form
a N-morpholino group; or where (2) RIII, R V, RV and N
taken together, may represent quinoline, isoquinoline
or hexamethylene tetramine.
The following examples exemplify the second
procedure:
Example 5 -
522 grams of morpholine (6 moles) were cooled to : -
20C and 125 grams of 1,4-dichloro-2-butene (1 mole) were
added dropwise with constant stirring and cooling to keep
the temperature at 50C - 60C. The entire addition took
about 1 hour, and stirring was continued for about one more
hour. While stirring, 150 grams of water was poured into
the reaction mixture, followed by 200 grams of 50~ sodium
hydroxide solution, then the mixture was allowed to separate.
-12-
~073~6~
The organic layer was removed, and the unreacted
morpholine was removed by distillation under reduced pressure.
The residue was washed with water and filtered, yielding a
yellow solid melting at 79C - 83C. This was 1,4-bis-
(N-morpholino)-2-butene. -
Since the purpose of the excess morpholine was to
act as an acid acceptor, the experiment was repeated, but
with 212 grams of sodium carbonate (2 moles) replacing the
excess 174 grams of morpholine (4 moles). The yield of
1,4-bis-(N-morpholino)-2-butene was about the same as the
previous synthesis. ~;
This reaction was repeated using 0.1 mole of
1,4-dichloro-2-butene and 0.6 mole of the following amines
in place of morpholine: piperidine, homopiperidine,
diethanolamine, dimethylamine, dipropylamine, dibutylamine,
di-(2-ethylhexyl) amine, dioctylamine, didecylamine,
didodecylamine, N-methyl propylamine, N-methyl butylamine,
N-methyl hexylamine, N-methyl octylamine, N-methyl
decylamine, N-methyl dodecylamine. All of these 1,4-bis-
amino-2-butenes were liquids, and were recovered from their
aqueous mixtures by partitioning.
Example 6
28.4 grams of 1,4-bis-dimethylamino-2-butene
(0.2 moles~ and 1.49 grams of triethanolamine (0.01 moles)
were dissolved in about 55.5 grams of water in a round-bottom
flask fitted with a s~irrer and reflux condenser, and 25.63
grams of 1,4-dichloro-2-butene (0.205 moles) were added
slowly while the mixture was stirred. The reaction mixture
was heated to 60 - 70C and maintained at that temperature,
-13-
1~73~6~ -
with stirring, for about 6 hours. The reaction was 98%
complete, as indicated by ionic chloride analysis. The
residue contained about 50% by weight of active material.
The procedure of Example 6 was repeated several
times using different proportions of reactants, as follows:
',
-14-
fO73~16~
~ _ In
~1k a~ o k
a)~ Q~ Q ~ k ~1
Q~ ~~ kt~
k~r ~a~ _I ~1
1~
XCO oer o _1 o er
W
~q bq U~
ou~ q k
~1k Q~ I e ,~ k
(~1 0~ O
~1~ ~Q~ k ~ k
co ~r
~;~r ~~ o o
.. .. ..
X CO o _I o o o o
W
Lg b~
~n
O ~ O
kb' k~' k S~ :
Q ~ ~
~r o~7 ~ 1`
~ ~ts~ -
Xco o. oOD O L~
W~--co--~-- ~D
_I~ Q~ k~ k
el~ ~
ou ~3 a~ ~-
1~
Xao o . o ~ o _I
-- U~
- - ~
t`k ~
a~s~ kh ~3 ~ k
Q~ ~ u~
~~ O C~
f~. .~, . . .
Xc~oo ~DO 1`
W~_~--~-- U7
a) :
~ .
R~
O
k
~ ~ ~ .
rl
k
O
,1 _i O
~ 0 ~1
R
- --I ~) --I 3
- 107346~
The procedure of Example 6 was again repeated,
except that the following reactants were used:
Example 12
A cf ~77efhl//4m/~ -
A 28.4 grams of 1,4-bis ~imcnth~lami~o-2-butene (0.2 moles)
1.94 grams of N-methyl morpholine (0.02 moles)
26.25 grams of 1,4-dichloro-2-butene (0.21 moles)
56.6 grams of water
Example 13
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles) --
2.00 grams of N-methyl piperidine (0.02 moles)
26.25 grams of 1,4-dichloro-2-butene ~0.21 moles)
56.7 grams of water
Example 14
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.06 grams of N-methyl homopiperidine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 molès)
56.7 grams of water
Example 15
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
1.7 grams of N-methyl pyrrolidine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles) -~
56.6 grams of water
Example 16
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.02 grams of butyldimethylamine (0.02 moles)
? 26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
56.9 grams of water
-16-
.. . . . . .
~73gG~
::;
Example 17
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.3 grams of pentyldimethylamine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
57.2 grams of water
Example 18
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.58 grams of hexyldimethylamine (0.02 moles)
26.5 grams ofl, 4-dichloro-2-butene (0.21 moles)
57.5 grams of water
Example 19
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.86 grams of heptyldimethylamine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
57.8 grams of water - -
Example 20 -
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
3.14 grams of octyldimethylamine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
58.1 grams of water
Example 21
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
3.42 grams of nonyldimethylamine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
58.3 grams of water
-17-
1~ ~ 6
Example 22
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
3.7 grams of decyldimethylamine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
58.6 grams of water
Example 23
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
3.98 grams of undecyldimethylamine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
58.9 grams of water -
Example 24
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
4.26 grams of dodecyldimethylamine (0.02 moles) ~
26.5 grams of 1,4-dichloro-2-butene (0.21 moles) ~ ~
59.2 grams of water ~ -
Example 25
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.7 grams of benzyldimethylamine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles) ~--
57.6 grams of water ~
Example 26 -
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.62 grams of quinoline (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
57.5 grams of water
-18-
.. . . . . .
iO73
Example 27
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.62 grams of isoquinoline (0.02 moles)
26,5 grams of 1,4-dichloro-2-butene (0.21 moles)
57.5 grams of water
Example 28
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
2.8 grams of hexamethylenetetramine (0.02 moles)
26.5 grams of 1,4-dichloro-2-butene (0.21 moles)
57.7 grams of water
Although in Examples 6 to 28 above, the difunc-
tional tertiary amine 1,4-bis-dimethylamino-2-butene was -~
utilized, any of the other difunctional tertiary amines,
as disclosed in Example 5, may be substituted in equivalent
molar amounts. Illustrative of such other difunctional
amines are, for example, 1,4-bis-(N-morpholino)-2-butene;
1,4-N,N'-dimethylpiperamine; 1,4-diazabicyclo (2.2.2)
octane; N,N,N', N'-tetramethylene diamine; N,N,N',N'-
tetra-(2-hydroxylpropyl)-ethylene diamine; 1,3-bis- -
(dimethylamino)-2-hydroxypropane; and 1,4-di-(N-homopiperidino)-
2-butene.
Furthermore, although only 1,4-dichloro-2-butene ~ -
has been illustrated above, 1,4-dibromo or 1,4-diiodo-2- -
butene may be substitued.
Example 29
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles) ;-
1.01 grams of butyldimethylamine (0.01 moles)
25.63 grams of 1,4-dichloro-2-butene (0.205 moles)
55.04 grams of water
--19--
.
.
~ ~ .
1073~6~
Example 30
28.4 grams of 1,4-bis-dimethylamino-2-butene (0.2 moles)
0.97 grams of N-methyl morpholine (0.01 moles)
25.63 grams of 1,4-dichloro-2-butene (0.205 moles)
55.0 grams of water
Example 31
The "Broth Dilution Test" method was used to
assay the antimicrobial properties of the products. In -~this method, 1.0 ml. of a solution of the material being
tested was added to 9.0 ml. of a suitable broth culture
medium in a test tube. A series of such test tubes was
prepared so that there was presented a stepwise decrease
in the concentrations being tested, from 0.1~ (1000 ppm)
to 0.005% (50 ppm). Each tube was then inoculated with
0.1 ml. of either a 24-hour broth culture of test bacteria,
or a 14-day aqueous spore suspension of test fungi. The -~
testing program was designed so that every concentration ~ --
of every material was inoculated separately by each
organism used in the test.
The test organisms employed were: ~
Escherchia coli (E. c.)
Pseudomonas seruginosa (Ps. a.)
Staphylococcus aureus (S. a.)
Streptococcus faecalis (S. f.)
Aspergillus niger (A. n.)
Penicillium expansum (P. e.)
After inoculation, the tubes were incubated as
follows:
-20-
`` 107:~46~
72 hours at 37C for bacteria
14 days at 28C for fungi
Following incubation, the tubes were examined for
the presence or absence of macroscopic growth as evidenced
by the presence or absence of turbidity.
The lowest concentration of the material being
tested at which macroscopic growth was not evident was de-
signated as the "Minimum Inhibitory Level" (M.I.L.)
Table 3, following shows the M.I.L. of the pro- -
ducts that were tested. The inhibitory concentrations are
shown in parts per million.
Table 3
Product Synthesized Gram Positive Gram Negative Fungi
in Example No. E.c. Ps.a. S.a.S.f. A n. P.e.
10001000 10001000 ~1000 ~1000
6 50 50 5050 71000 ~1000
11 50 50 5050 71000 ~1000
28 10001000 10001000 ~1000 71000
5050 ~1000 ~1000
20 27 100 500 100100 ~1000 ?1000
19 .1000100010001000 ~1000 ~1000
21 10001000 10001000 71000 ~1000
The results of these tests show that every com-
pound tested has anti-bacterial properties in concentrations
as low as 0.1%, and even lower in many cases.
Example 32
In order to estimate the bactericidal power of
these compounds, the product prepared in Example 7 was
assayed using the "~ater Treatment Test".
- -
lOq~
The product was dissolved in sterile distilled water
and diluted to the test concentration. Then 50 ml. of test
solution was added aseptically to previously sterilized cotton-
stoppered 125 ml. Erlenmeyer flasks. One set of flasks contai-
ning the product at concentrations of 25 ppm, 50 ppm, 100 ppm,
150 ppm, 200 ppm, 250 ppm, and 300 ppm, was inoculated by
introducing into each flask 0.5 ml. of a 1/10 nutrient broth
dilution of a 24-hour nutrient broth culture of Aerobacter
aerogenes. Another set of flasks containing the product at
the same concentrations was inoculated by introducing into
each flask 0.5 ml. of a 1/10 nutrient broth dilution of a 24-
hour nutrient broth culture of Pseudomonas aeruginosa.
At intervals of 30, 60 and 180 minutes following
inoculation of 1.0 ml. aliguot was withdrawn from each flask
and added to 9.0 ml. of sterile azolectin/"Tween 80" ("Tween
80" is a trade mark) neutralizer from which additional ten-
`fold serial dilutions were prepared in sterile neutralizer
solution.
~ .
~-Nutrient agar plates were prepared from 1 x 10
and 1 x 10 3 dilutions.
Simultaneously with each set of flasks, a control
of sterile distilled water was similarly inoculated and ali-
quots were taken at the same time intervals at 1 x 10 4, -
1 x 10 5 and 1 x 10 6 dilutions.
For additional control purposes, and also for com-
parison purposes, simultaneous assays were performed with
each set of flasks on "BTC 776" (trade mark), a powerful bac-
tericidal agent used in water treatment, the chemical name of
which is alkyl benzyl dimethyl ammonium chloride. It is manu-
- . -, ~:. , -, " : ... . .
`` ~07346~
factured and sold by Qnyx Chemical Co., of Jersey City, N.J.
Table 4 shows the number ofsur,v,ivorsof Aerobacter
aerogenes and Table 5 shows the number of survivors of ^^'-
Pseudomonas aeruginosa ATCC 15442, following exposure at
different concentrations for the indicated periods of time.
The numbers in the table must be multiplied by 1 x 102.
Concentrations of the materials being tested are given in ,-
parts per million.
Table 4
', ' .
CompoundConcentration No. of Surviving Bacteria per ml.
(Example No.) (ppm) (x 102) After
30 Min. 60 Min. 180 Min.
7 5 78 43 3
89 45 1 ,
3~ 19 0
59 15 0
47 12 0
37 14 0
57 7 0 '~'
23 1.5 0
27 3 0
22 4 0 ,'
2 0
21 3 0
21 3 0 " ~,
16 3 0
0 0 0
0 0 0
0 0 0
0 20,900 26,500 28,700
-23-
- : . - .. ...
" 1073~69~
Table 4 (continued)
CompoundConcentration No. of Surviving Bacteria per ml.
(Example No.) (ppm) (x 102) After
30 Min. 60 Min. 180 Min.
BTC (Control) 10 49 10 0
37 8 0
32 10 0 ~ '~
43 14 0
1 0 0
2 0 0
6 0 0
4 0 0
Table 5
Pseudomonas Aeroginosa
CompoundConcentration No. o~ Surviving Bacteria per ml. ~:
(Example No.~ (ppm) (x 10 ) After
30 Min. 60 Min. 180 Min.
`: -
7 20 9 0 0
1 0
13 0 0
1 0 0
0 0 0
0 92,500 91,500 91,000
BTC (Control) 10 1,260 180 0
1,380 123 0
1,460 118 0
1,450 192 0
214 16 0
235 18 0
208 57 0
137 15 0
145 18 0
162 17 0
-24-
~0734 ~
The compounds of this invention are all very soluble
in water and insoluble in organic solvents such as isopropanol,
acetone, hexane, trichloroethane, toluene, and the like.
They are, furthermore, non-foaming, which is an important
property because it makes these compounds suitable for use
as anti-microbial agents in products and processes where the
generation of foam would be undesirable.
In addition to their utilization as anti-microbal agents,
the compounds of this invention are also utilizable as hair
conditioning agents in shampoos and the like.
.-..
