Synthesis and anti-microbial activities of choline-like quaternary ammonium chlorides

Article abstract of DOI:10.1016/j.ejmech.2003.09.004

New choline-like quaternary ammonium chlorides were obtained. The work-up procedure of synthesis was quick and efficient. The obtained chlorides showed anti-microbial activities. Quaternary ammonium chlorides derivatives of deanol esters exhibited strong

Full text of DOI:10.1016/j.ejmech.2003.09.004

European Journal of Medicinal Chemistry 38 (2003) 1035ꢀ1042
/
www.elsevier.com/locate/ejmech
Short communication
Synthesis and anti-microbial activities of choline-like
quaternary ammonium chlorides
Juliusz Pernak *, Przemys law Chwa la
Poznan´ University of Technology, pl. M. Sk lodowskiej-Curie 2, 60-965 Poznan´, Poland
Received 13 February 2003; received in revised form 17 September 2003; accepted 17 September 2003
Abstract
New choline-like quaternary ammonium chlorides were obtained. The work-up procedure of synthesis was quick and efficient.
The obtained chlorides showed anti-microbial activities. Quaternary ammonium chlorides derivatives of deanol esters exhibited
strong activity and wide anti-bacterial spectra, similar to the activity of benzalkonium chloride. The relationship between chemical
structure and anti-microbial activity was analyzed by the QSAR method.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Quaternary ammonium chlorides; Deanol; Anti-microbial activities; QSAR analysis
1. Introduction
wood preservatives. In the preservation of wood, studies
of Butcher et al. [7,8] proved to be of key importance. In
the eighties, the compounds were applied as asphalt
modifiers and in the nineties as clay modifiers. At the
end of 20th century, specifically beginning from 1998 the
group of discussed compounds containing BF4ꢁ, PF6ꢁ,
(CF3SO2)2Nꢁ, etc. as anions have been named ionic
liquids (ILs). The subject of ionic liquids is well summed
Quaternary ammonium salts (quats) have been
known for over 100 years. They were obtained for the
first time by Menschutkin [1] in the reaction of tertiary
amine with alkyl halides early in 1890. As compared to
later described procedures, this way of the synthesis,
termed the Menschutkin reaction, has definitely been
more advantageous (irreversible reaction, high effi-
ciency). Practical application of quats has been rapidly
up in appropriate reviews [9ꢀ15]. The historic outline
/
presented above points to the important role played by
quats in industry. They consist of a cation of organic
type and an inorganic or organic anion. The number of
potential cation/anion combinations is very high and is
estimated at 1018. Nevertheless, no more than 500 quats
are commercially available at present. Mass application
of quats has its positive and negative aspects. The
positives include their polyfunctionality. In commercial
preparations, such compounds may fulfil several func-
tions in parallel, exerting surface activity, anti-microbial
and antielectrostatic activity. Compounds of this type
will be of particular interest in 21st century. Develop-
ment of resistance in microbes and the relatively slow
decomposition in the natural environment belong to the
most important negative aspects of quats application
[16,17]. New quats may supplement the presently
applied ones, to which microbes developed resistance.
The mechanism of quats action remains the same. The
developing during the 20th century. In 1915ꢀ16 Jacobs
/
and coworkers [2,3] described the bactericidal activity
but they were not used for disinfection until 1935,
following publication of the study by Domagk [4].
Following the latter publication, the investigators’
attention was drawn to the surface activity of quats
containing hydrophobic group of more than 8 carbon
atoms. In the sixties, the compounds were found to be
excellent fabric softeners. In 1965 Makosza and Serafi-
˛
nowa [5] described catalytic activity of the group of
compounds. The reactions, in which quats are used as
catalysers, are termed the JarousseꢀMakosza reaction
/
˛
[6] or phase transfer catalysis. In the seventies the
compounds were used as antielectrostatic agents and
* Corresponding author.
E-mail address: juliusz.pernak@put.poznan.pl (J. Pernak).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.09.004

1036
J. Pernak, P. Chwa la / European Journal of Medicinal Chemistry 38 (2003) 1035ꢀ1042
/
search for new, effective compounds continues [18ꢀ
We have focused our attention on new derivatives of
choline.
/
27].
microbial activity [18,24,25]. Therefore all synthesized
quats were tested in vivo for their reaction against cocci,
rods, fungi and bacilli. The minimum inhibitory con-
centration (MIC) series of compounds were determi-
nated by tube dilution method.
2. Chemistry
3.2. QSAR analysis
We prepared five new groups of ammonium chlorides
which originated from the following aminoalcohols: 2-
dimethylaminethanol (deanol) (1), 3-dimethylamino-1-
propanol (2), 1-dimethylamino-2-propanol (3), and
aminoesters: 2-(dimethylamino)ethyl decanate (4), 2-
(dimethylamino)ethyl benzoate (5). The quaternary
ammonium choline-like derivatives based on deanol
structure were obtained by nucleophilic substitution
Anti-microbial activity of quats remained, among
other factors, lipophilicity-dependent. Good approxima-
tion for compound lipophilicity was provided by 1-
octanol/water partition coefficient (P). Therefore, the
QSAR analysis was constructed mostly using a simpli-
fied Hansch equation:
log RBRꢃaꢂb×Clog Pꢂc×Clog P2
with chloromethyl alkyl ethers, containing 3ꢀ
/18 carbons
The RBR (relative biological response) parameter used
to correspond to 1/C, where C was minimal inhibitory
concentration (MIC). The logarithm of 1-octanol/water
in alkoxy groups, in hexane.
partition coefficient was calculated using the Meylanꢀ
/
Howard method [28].
All of the compounds were new and obtained in high
yields (Table 1). The structure of new compounds was
established by 1H-NMR and 13C-NMR or elemental
analysis. The results of elemental analysis were in
agreement with the theoretical values. Generally, the
most important signals, characteristic for the chlorides
4. Results and discussion
New choline-like quaternary ammonium derivatives
of 6ꢀ
/
10, listed in Table 1, were synthesized by
5 with
Menschutkin reaction. Quaternisation of 1ꢀ
/
chloromethyl alkyl ethers was quick and efficient. The
mechanism of the reaction was SN1, in which formation
of the cation R1OCH2ꢂ by ionization proved to be the
slowest step. Disadvantages of chloromethyl alkyl ethers
involved its very easy hydrolysis in the presence of traces
of moisture. Therefore, quaternisation had to be con-
ducted under strictly anhydrous conditions.
came from protons of NꢂCH2O, which in chlorides 6ꢀ
were observed as a singlet at 4.9ꢀ5.0 ppm and in 9 and
10 as a singlet at 5.1ꢀ
5.2 ppm. In the 13C-NMR spectra,
the chemical shift of the carbon of NꢂCH2O appeared
at 90ꢀ91 ppm.
/
8
/
/
/
All of the obtained compounds proved to be strongly
hygroscopic. In the extreme cases they were left in a
crystallizer and after a few minutes they formed a water
solution. Absorption of water from the surrounding
atmosphere was intense and rapid. The synthesized salts
could be estimated by a direct two-phase back titration
(EN ISO 2871-1). We established that for chlorides 7, 9
and 10 the alkoxy group had to contain at least seven
carbon atoms, for chlorides 8 eight carbon atoms and
for chlorides 6 ten carbon atoms. The percentages of
chloride in the products are listed in Table 1. The
remaining fraction (up to 100%) was formed by water.
Despite the restricted contact with water, the product
contained significant amounts of water.
Generally, alcohol derivatives of quaternary ammo-
nium chlorides 6ꢀ
chloroform and insoluble in hexane for 6ꢀ
soluble in hot hexane for 9. The water solutions of
chlorides 6ꢀ10 were stable at room temperature.
Minimal inhibitory concentration (MIC) values, de-
termined for prepared chlorides 6ꢀ10, are given in
Tables 2ꢀ6. All studied compounds were active against
/10 were well soluble in water and
/
8 and 10, but
/
/
/
the microorganisms. Activities of investigated com-
pounds were distinctly higher for Gram-positive bac-
teria than for Gram-negative bacteria. Similarly to
previous literature data [24], the highest resistance
against the compounds was exhibited by Pseudomonas
aeruginosa. Surprisingly, the activity against fungi was
similar to that against Gram-positive bacteria.
3. Pharmacology
Structure of the molecules permitted us the activity of
the examined chlorides. The obtained chlorides could be
divided to two groups: alcohol quartenary derivatives
3.1. Anti-microbial activity
(6aꢀ
/
8j) and ester quartenary derivatives (9aꢀ10j) with
/
The quaternary ammonium chlorides with alkoxy-
methyl substituent in many cases demonstrated anti-
large substituents. In general, MIC values of the ester
derivatives were slightly lower than those of alcohols.

J. Pernak, P. Chwa la / European Journal of Medicinal Chemistry 38 (2003) 1035ꢀ
/1042
1037
Table 1
Quaternary ammonium chloride (6ꢀ
/
10) prepared
Chloride
R1
R2
Melting point (8C)
Yield (%)
Purity (%)
6a
6b
6c
6d
6e
6f
C6H13
C7H15
C8H17
C9H19
HOCH2
HOCH2
oil
90.5
89
94
86
88
87.5
88
90
87
85
90
88
89
85
86
87
92
89
92
91
83.5
81
87
85.5
87
84
82
85
84
85
85
81
79
85
88
83
85.5
87
85
87
88
89
87
86
87.5
88
89
85
87
80
ꢀ
ꢀ/
ꢀ/
92
/
hygroscopic
hygroscopic
49-51
HOCH2
HOCH2
C
10H21
C11H23
12H25
C14H29
16H31
HOCH2
HOCH2
66.5ꢀ
68.5ꢀ
69.5ꢀ
60ꢀ
71ꢀ
85ꢀ
oil
/
68.5
97
/70
98.5
99.5
99
6g
6h
6i
C
HOCH2
HOCH2
HOCH2
/71.5
/
63
C
/
73.5
99
6j
C18H35
C6H13
C7H15
C8H17
C9H19
C10H21
HOCH2
/
87
97
7a
7b
7c
7d
7e
7f
HOCH2CH2
HOCH2CH2
HOCH2CH2
HOCH2CH2
HOCH2CH2
HOCH2CH2
HOCH2CH2
HOCH2CH2
HOCH2CH2
HOCH2CH2
HO(CH3)CH
HO(CH3)CH
HO(CH3)CH
HO(CH3)CH
HO(CH3)CH
HO(CH3)CH
HO(CH3)CH
HO(CH3)CH
HO(CH3)CH
HO(CH3)CH
C9H19COOCH2
C9H19COOCH2
C9H19COOCH2
C9H19COOCH2
C9H19COOCH2
C9H19COOCH2
C9H19COOCH2
C9H19COOCH2
C9H19COOCH2
C9H19COOCH2
PhCOOCH2
PhCOOCH2
PhCOOCH2
PhCOOCH2
PhCOOCH2
PhCOOCH2
PhCOOCH2
PhCOOCH2
PhCOOCH2
PhCOOCH2
ꢀ/
88
92
hygroscopic
hygroscopic
hygroscopic
94
63.5ꢀ
66.5ꢀ
/66
96.5
96
C
11H23
C12H25
14H29
C16H31
18H35
/69
7g
7h
7i
69ꢀ
73ꢀ
83ꢀ
89ꢀ
oil
oil
oil
oil
/
71
76
85
91
98
C
/
97.5
98
/
7j
C
/
97
8a
8b
8c
8d
8e
8f
C6H13
C7H15
C8H17
C9H19
C10H21
C11H23
C12H25
ꢀ/
ꢀ/
81
86
90
91
95
96
96.5
95
hygroscopic
hygroscopic
8g
8h
8i
35ꢀ
41ꢀ
44ꢀ
48ꢀ
oil
oil
/
38
44
47
51
C
14H29
/
C16H31
C18H35
C3H7
/
8j
/
8a
9b
9c
9d
9e
9f
ꢀ/
ꢀ/
ꢀ/
ꢀ/
95
95
C4H9
C5H11
C6H13
C7H15
C8H17
C9H19
hygroscopic
hygroscopic
50ꢀ
57ꢀ
62.5ꢀ
65ꢀ
67ꢀ
75ꢀ
oil
oil
/
54
/
60
9g
9h
9i
/64
99
99
C
10H21
C11H23
12H25
/
67
69
76
/
99
9j
C
/
99.5
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
C6H13
C7H15
C8H17
C9H19
C10H21
C11H23
ꢀ/
83
86
89
95
98
98
99
98.5
97
hygroscopic
hygroscopic
54ꢀ
71ꢀ
76ꢀ
84ꢀ
86ꢀ
87ꢀ
/
58
74
79
86
88
90
/
C
12H25
C14H29
16H31
C18H35
/
/
C
/
/
On the other hand, in the studies, the length of
alkoxymethyl group exerted a more pronounced influ-
ence on bacteriostatic activity. The most favorable
MIC values of 9f, 9g and 10f, 10g was similar to the ones
of commercially available benzalkonium chloride
(BAC), especially for Gram-positive bacteria. The series
of quaternary 3-dimethylamino-1-propanol derivatives
alkoxymethyl group contained 9ꢀ
The highest activity in the series was revealed: 6g*
26.6ꢀ426.8 mM (7.8ꢀ 1.7ꢀ211.2 mM
125 mg Lꢁ1); 8h*
(0.49ꢀ 0.9ꢀ107.1 mM (0.25ꢀ31.25 mg
62.5 mg Lꢁ1); 9f*
Lꢁ1); 10g* 15.63 mg Lꢁ1). The
0.3ꢀ42.2 mM (0.125ꢀ
/14 carbon atoms.
/
(7aꢀ
number of carbons in alkoxy substituents, where 7d
showed maximum activity for the even set (1.1ꢀ269.6
mM or 0.25ꢀ
62.5 mg Lꢁ1), and 7h for the odd set (0.8ꢀ
/7j) can be considered as two sets: with an even
/
/
/
/
/
/
/
/
/
/
/
/
/
/

1038
J. Pernak, P. Chwa la / European Journal of Medicinal Chemistry 38 (2003) 1035ꢀ1042
/
Table 2
MIC values a and calculated 1-octanol/water partition coefficients (Clog P) of alkoxymethyl(2-hydroxyethyl)dimethylammonium chlorides (6aꢀ
6j)
/
b
Strain
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
BAC
P. aeruginosa
E. coli
ꢀ
/
2540
2540
2540
2540
2540
2540
2540
2540
2540
2540
ꢀ
/
2350
2350
2350
2350
2350
2350
2350
2350
2350
2350
2190
2190
2190
2190
2190
2190
2190
2190
2190
2190
2040
1020
1020
1020
510.7
510.7
510.7
510.7
510.7
510.7
958.6
239.6
479.3
479.3
239.6
239.6
239.6
239.6
239.6
239.6
451.5
112.9
225.8
225.8
112.9
112.9
112.9
225.8
112.9
112.9
426.8
106.7
213.4
213.4
53.4
809.2
101.2
202.3
404.6
101.2
202.3
202.3
101.2
101.2
202.3
1540
ꢀ
/
1470
1470
1470
1470
1470
733.1
1470
1470
1470
1470
23.0
5.7
ꢀ
/
769.3
769.3
769.3
384.6
192.3
384.6
384.6
192.3
384.6
ꢀ
/
K. pneumoniae
P. vulgaris
S. aureus
ꢀ
/
ꢀ
/
ꢀ
/
11.5
5.7
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ/
0.7
0.7
M. luteus
E. faecalis
C. albicans
R. rubrum
B. subtilis
Clog P
ꢀ
/
26.6
53.4
ꢀ
/
0.4
1.4
ꢀ
/
ꢀ
/
53.4
53.4
ꢀ
/
ꢀ
/
1.4
0.7
ꢀ/
ꢀ/
53.4
ꢀ/
ꢁ/1.67
ꢁ
/
1.18
ꢁ
/
0.68
ꢁ
/
0.19
0.30
0.79
1.28
2.26
3.24
4.23
3.32
a
In mM, the inoculum is 0.1 mL of suspension with the number of microorganisms ranged from 103 to 104.
BAC is benzalkonium chloride.
b
422.4 mM or 0.25ꢀ
/
125 mg Lꢁ1). The obtained values for
of quaternary 3-dimethylamino-1-propanol derivatives
(7aꢀ7j), the QSAR equation correlation coefficients
were high and fluctuated between 0.9903 to 0.9225.
The predicted value of equations ranged between 98 and
85%. The population of carbon atoms odd number
series of quaternary 3-dimethylamino-1-propanol deri-
vatives was too small for statistical processing. For ester
7 imply the possibility of a different kind of disruption
of the cell membrane for these two sets. These results
require further research.
/
Data of Figs. 1 and 2 demonstrate the dependency of
the average logarithm of relative biological response
values for all strains upon the alkoxymethyl chain
length: 6ꢀ
Results of QSAR analysis of the bacteriostatic
activity of prepared chlorides 6ꢀ10 is given in Table 7.
/
18 carbon atoms for 6ꢀ
/
8 and 10 or 3ꢀ
/
12 for 9.
quaternary derivatives (9ꢀ
correlation coefficients were slightly lower than for
alcohol quaternary chlorides (6ꢀ8).
/
10), the QSAR equation
/
/
The general equation was constructed based on the
Hansch equation for parabolic model relating, where
RBR was represented by 1/MIC of a tested compound
and MIC was the average value for all tested strains.
The results were presented in Table 7 as parameters of
regression equations with the appropriate statistical
data.
A statistically significant correlation was demon-
strated between the dependent variable: Clog P and
the structural parameter determined in this work. As
The statistically significant correlation of obtained
QSAR equations confirm that lipophlicity as Clog P is
the main factor of anti-microbial activity. The obtained
Clog Po parameters show that most active alcoholam-
monium chlorides 6ꢀ8 are between 1.60 and 2.61 and
/
for esterammonium chlorides: 6.55 for 9 and 5.85 for 10.
The comparison of QSAR parameters for these two
quat groups indicate that synthesis of chlorides with two
long chain substituents and more lipophilic cause
increasing anti-microbial activity.
regards alcohol quaternary derivatives (6aꢀ
/
7a, 7c, 7e,
Our research proves that choline-like quaternary
ammonium esters can be successfully used as anti-
7gꢀ
/
8j) but without the carbon atoms even number series
Table 3
MIC values a and calculated 1-octanol/water partition coefficients (Clog P) of alkoxymethyl(3-hydro-xypropyl)dimethylammonium chlorides (7aꢀ
7j)
/
b
Strain
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
BAC
P. aeruginosa
E. coli
ꢀ/
2720
2720
2720
2720
1361
680.5
680.5
1361
680.5
1361
ꢀ/
2500
1250
1250
2500
579.4
144.8
289.7
289.7
36.2
9.0
269.6
16.8
16.8
33.6
4.2
504.4
63.1
63.1
63.1
7.9
947.4
118.4
118.4
118.4
14.8
14.8
7.4
223.3
55.8
55.8
55.8
7.0
422.4
52.8
52.8
52.8
3.3
801.3
400.6
400.6
400.6
12.5
1520
762.1
762.1
762.1
23.8
23.0
5.7
K. pneumoniae
P. vulgaris
S. aureus
11.5
5.7
78.3
0.7
0.7
M. luteus
E. faecalis
C. albicans
R. rubrum
B. subtilis
Clog P
39.0
312.9
625.9
312.9
156.5
1.1
2.1
2.0
4.0
0.9
1.7
0.8
3.3
12.5
25.0
23.8
23.8
9.0
0.4
1.4
36.2
18.1
18.1
4.2
4.2
7.9
7.9
14.8
14.8
59.2
3.5
13.2
6.6
12.5
12.5
23.8
23.8
27.9
14.0
1.4
0.7
4.2
31.5
26.4
200.3
190.5
ꢁ
/
1.17
ꢁ/
0.68
ꢁ/0.19
0.30
0.79
1.28
1.77
2.75
3.74
4.72
3.32
a
In mM, the inoculum is 0.1 mL of suspension with the number of microorganisms ranged from 103 to 104.
BAC is benzalkonium chloride.
b

J. Pernak, P. Chwa la / European Journal of Medicinal Chemistry 38 (2003) 1035ꢀ
/1042
1039
Table 4
MIC values a and calculated 1-octanol/water partition coefficient parameters (Clog P) of alkoxymethyl(2-hydroxypropyl)dimethyloammonium
chlorides (8aꢀ8j)
/
b
Strain
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
BAC
P. aeruginosa
ꢀ
/
2720
2720
2720
2720
1360
1360
2720
2720
1360
1360
2500
1250
1250
2500
625.9
625.9
1250
1250
625.9
625.9
2320
1160
1160
1160
289.7
289.7
579.4
579.4
289.7
289.7
1080
539.3
539.3
539.3
67.4
1010
252.2
252.2
252.2
31.5
473.7
236.8
236.8
236.8
14.8
446.6
111.6
223.3
223.3
7.0
211.2
105.6
105.6
211.2
1.7
400.6
200.3
200.3
200.3
3.1
762.1
381.0
381.0
381.0
190.6
190.6
190.6
95.3
23.0
5.7
Escherichia coli
K.pneumoniae
P. vulgaris
S. aureus
11.5
5.7
0.7
0.7
M. luteus
134.8
134.8
269.6
134.8
134.8
126.1
63.1
59.2
59.2
27.9
27.9
55.8
27.9
27.9
6.6
6.6
25.0
3.1
E. faecalis
C. albicans
R. rubrum
B. subtilis
Clog P
0.4
1.4
126.1
63.1
63.1
118.4
29.6
59.2
13.2
13.2
6.6
12.5
6.2
47.6
190.6
1.4
0.7
12.5
ꢁ/1.25
ꢁ
/
0.76
ꢁ
/
0.27
0.22
0.72
1.21
1.70
2.68
3.66
4.64
3.32
a
In mM, the inoculum is 0.1 mL of suspension with the number of microorganisms ranged from 103 to 104.
BAC is benzalkonium chloride.
b
microbial agents. Moreover the QSAR equations ob-
NMR spectra were recorded on a Varian model XL
1
300 spectrometer at 300 MHz for H and 75 MHz for
tained can be useful in the search for new quaternary
ammonium chlorides with stronger anti-microbial activ-
ity, which could be applied as new potential disinfec-
tants.
13C at 20 8C with tetramethylsilane as internal reference.
Products were confirmed by elemental analyses with
error in the range of: C 9
/
0.30%; H 9
/
0.28%; N 9
/
0.35%. Melting points were measured on Buchi melting
¨
point B-540.
Benzalkonium chloride applied in the research repre-
5. Experimental
sented a mixture of alkyls C12H25 (Â/60%) and C14H29
(Â40%), available from Aldrich.
/
5.1. Chemistry
The product of quaternization reaction was controlled
by TLC on silcagel 60 where the mobile phase was 9:1
Chloromethyl alkyl ether was obtained by passing
hydrochloride gas through a mixture of formaldehyde
and alcohol. The purity of ethers was determined as a
crude product by alkalimetric method: 1 g crude product
chloroformꢀmethanol solution.
/
was added to 10 mL acetone at ꢁ
/
20 8C. HCl-substrate
5.1.1. General procedure for alkoxymethylation of 1ꢀ3
/
was then quickly neutralized with 0.2 M KOH in
MeOH. Hot water (50 mL) was added carefully. HCl
as a product of hydrolysis of chloromethyl alkyl ether
was neutralized with 0.2 M KOH in MeOH. The crude
To a stirred solution of dimethylaminoalkanol (0.05
mol) in anhydrous hexane (20 mL) the corresponding
chloromethyl alkyl ether (0.055 mol) in anhydrous
hexane (10 mL) was added dropwise. After 10 min the
product contained 85ꢀ/99% chloromethyl alkyl ethers.
precipitate or oil was washed with 3ꢄ10 mL dry
/
Table 5
MIC values a and calculated 1-octanol/water partition coefficient parameters (Clog P) of alkoxymethyl(2-decanoyloxyethyl)dimethylammonium
chlorides (9aꢀ9j)
/
b
Strain
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
BAC
P. aeruginosa
E. coli
ꢀ/
2360
2360
2360
2360
ꢀ/
2200
2200
2200
2200
2050
512.8
512.8
512.8
8.0
481.1
120.1
120.1
120.1
3.8
226.6
28.3
14.1
14.1
1.8
107.1
6.7
6.7
6.7
1.7
3.4
0.9
3.4
1.7
1.7
203.0
12.7
6.3
771.7
48.2
24.1
12.0
6.0
735.3
46.0
91.9
22.9
5.7
702.2
175.5
87.8
87.8
11.0
21.9
11.0
21.9
87.8
11.0
23.0
5.7
K. pneumoniae
P. vulgaris
S. aureus
M. luteus
E. faecalis
C. albicans
R. rubra
11.5
5.7
6.3
3.2
18.4
295.2
9.2
34.2
137.2
34.2
0.7
0.7
64.1
8.0
15.0
3.8
7.1
1.8
6.3
1.6
6.0
6.0
11.5
5.7
0.4
1.4
295.2
9.2
590.5
274.4
4.3
274.4
128.2
2.0
64.1
60.0
1.9
7.1
1.8
3.2
6.3
6.0
5.7
24.1
6.0
46.0
5.7
1.4
0.7
B. subtilis
Clog P
60.0
7.1
5.76
6.3
6.74
3.79
4.29
4.78
5.27
6.25
7.23
7.72
8.21
3.32
a
In mM, the inoculum is 0.1 mL of suspension with the number of microorganisms ranged from 103 to 104.
BAC is benzalkonium chloride.
b

1040
J. Pernak, P. Chwa la / European Journal of Medicinal Chemistry 38 (2003) 1035ꢀ1042
/
Table 6
MIC values a and calculated 1-octanol/water partition coefficient parameters (Clog P) of alkoxymethyl(2-benzoyloxyethyl)dimethylammonium
chlorides (10aꢀ10j)
/
b
Strain
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
BAC
P. aeruginosa
E. coli
1830
862.7
862.7
862.7
862.7
107.8
26.9
408.7
204.4
204.4
408.7
51.1
194.2
97.1
97.1
194.2
12.1
6.1
92.5
46.2
46.2
46.2
2.9
44.2
11.0
11.0
22.0
1.4
42.2
21.1
21.1
21.1
0.3
323.8
81.0
81.0
81.0
1.3
621.8
310.9
310.9
310.9
9.7
1200
597.9
597.9
597.9
18.6
23.0
5.7
913.2
913.2
1830
K.pneumoniae
P. vulgaris
S. aureus
11.5
5.7
228.3
114.2
228.3
456.6
228.3
913.2
0.7
0.7
M. luteus
E. faecalis
C. albicans
R. rubrum
B. subtilis
Clog P
12.8
51.1
2.9
1.4
2.8
0.3
0.7
1.3
2.5
4.8
9.7
18.6
37.4
107.8
215.7
107.8
431.3
24.2
48.6
24.2
12.1
11.5
23.1
11.5
5.8
0.4
1.4
102.2
51.1
25.5
5.5
2.8
2.6
2.6
10.1
10.1
10.1
19.4
19.4
77.7
37.4
37.4
1.4
0.7
2.8
5.25
2.6
5.74
149.5
2.79
3.29
3.78
4.27
4.76
6.72
7.71
8.69
3.32
a
In mM, the inoculum is 0.1 mL of suspension with the number of microorganisms ranged from 103 to 104.
BAC is benzalkonium chloride.
b
hexane. The solid products were filtered and then
recrystallised from acetone.
5.1.1.1. Dodecyloxymethyl(2-hydroxyethyl)-
1
dimethylammonium chloride (6g). H-NMR (CDCl3) d
ppmꢃ
2H), 3.85 (t, Jꢃ
3.34 (s, 6H), 1.63 (t, Jꢃ
(t, Jꢃ
6.8 Hz, 3H). 13C-NMR (CDCl3) d ppmꢃ
/
5.85 ( t, Jꢃ
6.6 Hz, 2H), 3.69 (t, Jꢃ
6.8 Hz, 2H), 1.26 (m, 18H), 0.88
91.1,
/
5.4 Hz, 1H), 4.99 (s, 2H), 4.08 (s,
/
/
4.8 Hz, 2H),
/
/
/
73.4, 62.6, 55.3, 48.1, 31.5, 29.3, 29.2, 29.1, 29.0, 25.4,
22.3, 13.7.
5.1.1.2. Dodecyloxymethyl(3-hydroxypropyl)-
1
dimethylammonium chloride (7g). H-NMR (CDCl3) d
ppmꢃ
4H), 3.25 (s, 6H), 2.05 (m, Jꢃ
6.9 Hz, 2H), 1.26 (m, 18H), 0.88 (t, Jꢃ
NMR (CDCl3) d ppmꢃ90.3, 73.5, 59.3, 58.1, 47.3,
/
4.93 (s, 2H), 3.85 (q, Jꢃ
5.5 Hz, 2H), 1,62 (t, Jꢃ
6,9 Hz, 3H). 13C-
/
6.6 Hz, 1H), 3.71 (dt,
/
/
Fig. 1. Relation between alkoxy chain length and average log 1/MIC
/
for 6, 8ꢀ10.
/
/
31.7, 29.4, 29.4, 29.3, 29.13, 29.09, 25.6, 25.5, 22.4, 13.9.
5.1.1.3. (2-Hydroxypropyl)-
dimethylundecyloxymethylammonium chloride (8f). H-
1
NMR (CDCl3) d ppmꢃ
(m, 1H), 3.84 (t, Jꢃ6.6 Hz, 2H), 3.48 (d, Jꢃ
2H), 3.37 (s, 3H), 3.34 (s, 3H), 1.29 (d, Jꢃ6.0 Hz, 3H),
1.62 (m, Jꢃ7.1 Hz, 2H), 1.26 (m, 16H), 0.88 (t, Jꢃ7.1
Hz, 3H). 13C-NMR (CDCl3) d ppmꢃ
91.2, 73.4, 66.3,
/
5.00 (q, Jꢃ/7.7 Hz, 2H), 4.49
/
/2.7 Hz,
/
/
/
/
61.3, 48.6, 48.2, 31.6, 29.4, 29.32, 29.26, 29.1, 25.6, 22.4,
22.0, 13.8.
5.1.2. General procedure for alkoxymethylation of 4 and
5
To stirred solution of aminoester (0.05 mol) in
anhydrous hexane (30 mL), the corresponding chloro-
methyl alkyl ether (0.06 mol) was added slowly. The
mixture was stirred for 10 min at 50 8C. The precipitate
Fig. 2. Relation between alkoxy chain length and average log 1/MIC
for 7.
or oil was washed with 3ꢄ10 mL dry hexane.
/

J. Pernak, P. Chwa la / European Journal of Medicinal Chemistry 38 (2003) 1035ꢀ
/1042
1041
Table 7
Slopes b, c and intercepts a of linear relationship between logarithm of reciprocal of average MIC against all tested microbial strains and
theoretically calculated logarithm of water-1-octanol partition coefficient for quaternary ammonium chlorides (6ꢀ10), Hansch equation form: log(1/
MIC)ꢃaꢂb ×Clog Pꢂc ×
Clog P2
/
/
/
/
/
/
Chlorides
a
b
C
r
r2
s
p
Clog Po
6aꢀ
7odd
/
6j
ꢁ
/
2.6690 (9
2.2567 (9
2.6513 (9
12.2694 (9
7.4396 (9
/
0.0600)
0.1005)
0.0310)
1.8398)
0.8321)
0.4880 (9
0.5846 (9
0.5289 (9
3.2871 (9
2.0963 (9
/
0.0456)
0.0869)
0.0299)
0.6364)
0.3140)
ꢁ
/
0.1528 (9
0.1472 (9
0.1013 (9
0.2511 (9
0.1792 (9
/
0.0151)
0.0221)
0.0080)
0.0527)
0.0272)
0.9720 0.9447 0.1499
0.9601 0.9219 0.1950
0.9903 0.9808 0.0793
0.9225 0.8510 0.2921
0.9297 0.8643 0.2694
B
/
0.0004
0.0061
0.0000
0.0013
0.0009
1.60
1.99
2.61
6.55
5.85
ꢁ
/
/
/
ꢁ
/
/
B
/
8aꢀ
/
8j
ꢁ
/
/
/
ꢁ
/
/
B
/
9aꢀ
/
9j
ꢁ
/
/
/
ꢁ
/
/
B
/
10aꢀ
/10j
ꢁ/
/
/
ꢁ/
/
B/
r denotes the correlation coefficient; r2 is the predicted value of equation; s is the standard error of estimation; p is significance level of regression
equations; numbers in parenthesis are S.D. of regression coefficients and Clog Po is the apex of the parabola.
The solid products were filtered and, than, recrystal-
Minimal inhibitory concentration (MIC) was deter-
mined by the tube dilution method. Twofold dilutions of
lized from hexaneꢀ
/
acetone system for 9aꢀ/9j and from
ethyl acetate for 10aꢀ
/
10j.
quats were prepared in MullerꢀHinton broth medium
/
¨
(bacteria) or Sabouraud broth medium (fungi). The
samples were incubated at 37 8C (bacteria) and at 26 8C
(fungi) and after 48 h the results were recorded. Growth
of the microorganisms was determined visually. The
lowest concentration at which there was no visible
growth (turbidity) was taken as the MIC.
5.1.2.1. (2-Decanoyloxyethyl)-
dodecyloxymethyldimethylammonium chloride (9g). H-
1
NMR (CDCl3) d ppmꢃ
Hz, 2H), 4.00 (t, Jꢃ4.7 Hz, 2H), 3.88 (t, Jꢃ
2H), 3.44 (s, 6H), 2.34 (t, Jꢃ7.7 Hz, 2H), 1.62 (dt, 4H),
1.265 (m, 24H), 0.88 (t, Jꢃ
6.9 Hz, 6H); 13C-NMR
(CDCl3) d ppmꢃ172.6, 90.9, 73.7, 59.3, 57.3, 47.9,
/
5.15 (s, 2H), 4.55 (t, Jꢃ
/
4.7
/
/6.6 Hz,
/
/
5.3. QSAR analysis
/
33.8, 31.6, 29.5, 29.3, 29.21, 29.16, 29.0, 28.9, 25.6, 24.5,
22.5, 13.9.
5.3.1. Structural parameters
The theoretically calculated logarithm of 1-octanol/
water partition coefficient parameters (Clog P) were
chosen as structural parameters for the obtained qua-
5.1.2.2. (2-Benzoyloxyethyl)-
dodecyloxymethyldimethylammonium chloride (10g).
ternary ammonium chlorides 6ꢀ10. For the quats, the
/
1H-NMR (CDCl3) d ppmꢃ
/
8.01 (d, Jꢃ
7.4 Hz, 1H), 7.45 (t, Jꢃ7.4 Hz, 2H), 5.22 (s,
2H), 4.83 (t, 2H), 4.20 (t, 2H), 3.84 (t, Jꢃ6.3 Hz, 2H),
3.52 (s, 6H), 1.55 (m, 2H), 1.25 (m, 18H), 0.88 (t, Jꢃ6.9
Hz, 3H). 13C-NMR (CDCl3) d ppmꢃ
165.2, 133.4,
/
7.4 Hz, 2H),
parameters (Clog P) were calculated by KOWINTM v.1.66
program (Syracuse Research Corporation, New York,
NY) from software package EPI SuiteTM v.3.10 (U.S.
Environmental Protection Agency), which is based on
7.60 (t, Jꢃ
/
/
/
/
/
the MeylanꢀHoward method.
/
129.5, 128.4, 128.4, 90.7, 73.6, 59.3, 58.1, 48.0, 31.7,
29.46, 29.43, 29.2, 25.6.
5.3.2. Statistics
The statistical analysis was carried out using Statistica
v.6 software (StatSoft, Inc., Tulsa, OK). In the multi-
variate regression analysis, the tested microorganisms
average minimal inhibitory concentration value for
5.2. Anti-microbial activity
The following microbial strains were used as test
organisms: Gram-negative rods; Pseudomonas aerugi-
nosa NCTC 6749, Escherichia coli ATCC 25922,
Klebsiella pneumoniae var. pneumoniae ATCC 33495,
Proteus vulgaris NCTC 4635, Gram-positive cocci;
Staphylococcus aureus NCTC 4163, Micrococcus luteus
NCTC 7743, Enterococcus faecalis ATCC 29212, Gram-
positive bacillus; Bacillus subtilis ATCC 6633, and
yeast-like fungi; Candida albicans ATCC 10231, Rhodo-
torula rubra (Demml 1889, Lodder 1934). Standard
strains were supplied by National Collection of Type
Cultures (NCTC), London and American Type Culture
Collection (ATCC). The R. rubra was obtained from the
Department of Pharmaceutical Bacteriology, Poznan´,
Poland.
chlorides 6ꢀ10 was related to structural parameters
/
(independent variables).
Acknowledgements
We are grateful for the financial support received
from the Polish Committee for Scientific Research
DS32/007/2003.
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