Synthesis and antimicrobial activity of a series of optically active quaternary ammonium salts derived from phenylalanine

Article abstract of DOI:10.2478/s11532-009-0126-8

We synthesized nine quaternary ammonium compounds (QUATs) starting from phenylalanine, N-alkyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium bromides, which were prepared as optically pure substances. Five compounds were prepared as S-enantiomers and four compounds as R-enantiomers. These compounds were evaluated by their activities against bacteria and fungi. Three microbial strains were used in the study: the gram-negative bacteria Escherichia coli, the gram-positive bacteria Staphylococcus aureus and the fungi Candida albicans. The activities were expressed as minimum bactericidal or fungicidal concentrations (MBC). The most active compounds were (2S)-N-tetradecyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium bromide and (2R)-N-tetradecyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium bromide, with MBC values exceeding those of commercial benzalkoniumbromide (BAB) used as standard. The relationships between structure and biological activity of the tested QUATs were quantified by the bilinear model (QSAR) and are discussed. Versita Warsaw and Springer-Verlag Berlin Heidelberg.

Full text of DOI:10.2478/s11532-009-0126-8

Cent. Eur. J. Chem. • 8(1) • 2010 • 194–201
DOI: 10.2478/s11532-009-0126-8
Central European Journal of Chemistry
Synthesis and antimicrobial activity of a series
of optically active quaternary ammonium salts
derived from phenylalanine
Research Article
Miloš Lukáč1*, Ivan Lacko1, Marián Bukovský2, Zuzana Kyselová1, Janka Karlovská3, Branislav
Horváth4, Ferdinand Devínsky1
1Department of Chemical Theory of Drugs, Faculty of Pharmacy,
Comenius University, Bratislava 832 32, Slovakia
2Department of Cellular and Molecular Biology of Drugs, Faculty of Pharmacy,
Comenius University, Bratislava 832 32, Slovakia
3Department of Physical Chemistry of Drugs, Faculty of Pharmacy,
Comenius University, Bratislava 832 32, Slovakia
4Institute of Chemistry, Faculty of Natural Science,
Comenius University, Bratislava 842 15, Slovakia
Received 16 April 2009; Accepted 31 August 2009
Abstract: We synthesized nine quaternary ammonium compounds (QUATs) starting from phenylalanine, N-alkyl-N,N-dimethyl-(1-hydroxy-
3-phenylpropyl)-2-ammonium bromides, which were prepared as optically pure substances. Five compounds were prepared as
S-enantiomers and four compounds as R-enantiomers. These compounds were evaluated by their activities against bacteria and fungi.
Three microbial strains were used in the study: the gram-negative bacteria Escherichia coli, the gram-positive bacteria Staphylococcus
aureus and the fungi Candida albicans. The activities were expressed as minimum bactericidal or fungicidal concentrations (MBC).
The most active compounds were (2S)-N-tetradecyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium bromide and (2R)-
N-tetradecyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium bromide, with MBC values exceeding those of commercial
benzalkoniumbromide (BAB) used as standard. The relationships between structure and biological activity of the tested QUATs were
quantified by the bilinear model (QSAR) and are discussed.
Keywords: Microbicidal activity • Enantiomers • Phenylalanine • Quaternary ammonium compounds • Quantitative structure activity relationships
(QSAR)
© Versita Warsaw and Springer-Verlag Berlin Heidelberg.
environment for a long time. Some of the quaternary
ammonium compounds (QUATs) fulfill these conditions
1. Introduction
[3]. The mode of the antimicrobial action of QUATs
lies in their interaction with cytoplasmic membrane of
bacteria or fungi. QUATs show a generalized damage
of cytoplasmic membrane so that the positively
charged “head” of the molecule interacts with the
negatively charged membrane components followed
by penetration of the non-polar tenside constituent into
its hydrophobic part. The crucial first step of membrane
destruction is the reduction of its electrical potential
by Coulombic interactions with the ammonium head
For a long time, positively charged compounds such
as quaternary ammonium salt derivatives have been
widely used as disinfectants in medicine, agriculture and
industry.As more and more resistant organisms continue
to emerge in the environment, identification of additional
antimicrobial agents is becoming increasingly important
[1,2]. It is expected that ideal antimicrobial agents for
wide use should fulfill some of the following conditions.
They have to possess strong antibacterial activity, be
safe in relation to humans, and should not persist in the
* E-mail: lukac@fpharm.uniba.sk
194

M. Lukáč et al.
[4]. The “neutralization” of the negative charge of the kinetic effects, interaction with proteins or lipid bilayers
membrane and interactions with the lipidic constituents [18,19]. QUATs are not only active against diseases
with the hydrophobic tail of the molecule causes changes causedbybacteriaorfungi,butalsoexhibitantiprotozoal
in the architecture and consequently in the fluidity of the [20,21], antineoplastic [22-24] and immunomodulatory
membrane. Finally, the membrane loses its permeability, activity [25]. They act as neuromuscular relaxants [26],
leakage follows and the intracellular constituents are as selective inhibitors of Aβ fibril formation [27], or as
released from the cell [4-8]. Other types of surfactants like antagoniststoneuronalnicotinicacetylcholinereceptors
nonionic or anionic detergents can increase susceptibility mediating dopamine release [28,29]. Recently QUATs
to QUATs via increasing membrane permeability and polycationic compounds have been intensively
(Escherichia coli) or they can reduce susceptibility to investigated as possible non-viral vectors for gene
QUATs by lowering cell surface hydrophobicity (Listeria transfection [30-33].
monocytogenes) [9]. Bjergbæk et al. investigated the
QUATs derived from phenylalanine possess
effect of oxygen limitation, glucose-starvation and interesting physico-chemical properties. Diego-
temperature on the susceptibility of Escherichia coli Castro et al. used (2S)-N-hexadecyl-N,N-dimethyl-
towards the quaternary ammonium biocide benzalkonium (1-hydroxy-3-phenylpropyl)-2-ammonium
chloride
chloride. They discovered that increasing temperatures, and (2S)-N-hexadecyl-N,N-dimethyl-(1-hydroxy-3-
the absence of oxygen, and energy substrates increased phenylpropyl)-2-ammonium bromide to study the
the antimicrobial effect of benzalkonium chloride towards enantioselective Diels-Alder cycloaddition reaction
E. coli [10]. Marcotte et al. investigated the influence with cyclopentadiene and nonyl acrylate [34]. Our
of the QUATs head group and alkyl chain with different laboratory has also synthesized some QUATs
length on their permeability-perturbing power and on derivatives of phenylalanine. It was found that racemic
their affinity for lipidic membranes. Three series of (3-dodecanoyloxy-1-phenylpropane-2-yl)trimethyl-
QUATs were used: benzalkonium, alkylpyridinium and ammonium bromide exhibits lower critical micelle
tetraalkylammonium derivates. They found that QUATs concentration than its S-enantiomer [35]. In the last
bearing C16 long chain were more efficient in decreasing few years homochiral QUATs derived from other amino
the membrane permeability than their C12 analogues. In acids have also been synthesized [36-39].
contrast, the chemical nature of the ammonium head
Although the mode of action of QUATs is by
group had practically no influence on the permeability destruction of the bacteria cell membrane(s), some
perturbations caused by QUATs bearing C16 chains [11]. fine structural features e.g. optical activity could have
The antimicrobial activity of QUATs is closely related to some influence on processes playing a crucial role
their surfactant properties [12-15]. The common structure during the inactivation of microorganism. If the QUATs
of QUATs is a long alkyl chain attached to a polar “head- are able to act via other mechanisms than nonspecific
group” that is formed by the ammonium cation portion destruction of membrane, e.g. action via enzymes,
of the molecule. Structure-activity relationship studies on they will have important roles in antimicrobial activity.
a variety of synthetic QUATs showed that the long alkyl Therefore we prepared and characterized a series
chain and ammonium cation may represent the minimal of some novel optically pure active QUATs (R and S
structural requirements for sufficient antimicrobial effects. isomers) and tested their antimicrobial activity against
Optimal antimicrobial activity is achieved at lengths of different representatives of microorganisms (G- and G+
the alkyl chain ranging from 10 to 18 carbons [16]. The bacteria, fungi). The starting material for the synthesis
correlation between critical micelle concentration (cmc) of bioactive compounds was phenylalanine.
and minimum inhibitory concentration (MIC) as a function
of alkyl chain length show that compounds of the QUAT
.
.
type with cmc in range 1 10-2 to 1 10-4 mol.dm-3 exhibit the
2. Experimental procedure
best antimicrobial activity [17]. In a homologous series of
QUATs, the antimicrobial activity increases progressively 2.1. Materials and methods
with increasing chain length up to a critical point beyond All chemicals used for synthesis were purchased
which the activity decreases. This behaviour is called the from commercial suppliers. BAB was obtained from
cut-off effect. It is a general feature of many of biologically Fluka, GmbH. Blood agar base No.2, Sabouraud
active compounds, especially those with a repeating agar, Nutrien broth No.2 and Sabouraud medium
structural motive such as a long hydrophobic alkyl chain were purchased from Imuna Pharm a.s. Slovakia. The
(e.g. alcohols, QUATs). There are many reasons for the elemental analyses of C, H and N were performed
existence of a cut-off in the biological activity of a given using a Carlo Erba analyser. Melting points of the
surfactant. These include limited aqueous solubility, compounds synthesized were determined using a
195
195

Synthesis and antimicrobial activity of a series of optically active
quaternary ammonium salts derived from phenylalanine
Kofler Micro Hot Stage and were uncorrected. NMR positive bacteria Staphylococcus aureus ATCC 6538
spectra were recorded on a Varian Gemini 2000 and fungi Candida albicans CCM 8186. The minimum
spectrometer at 300 MHz for 1H and 75 MHz for 13C at bactericidal or fungicidal concentration of compounds
20°C with tetramethylsilane used as internal standard. 5S-9S, 5R-8R and rac-5–rac-8 was determined by the
Infrared spectra were recorded on a FT-IR Impact 400 D modified method described in [41].
spectrophotometer as potassium bromide discs. Optical
Solutions of compounds studied, 5S-9S, 5R-8R,
rotations of compounds were measured in ethanol rac-5–rac-8 and BAB were prepared in water. A
solution c = 1.1 on a JASCO P1010 polarimeter. The suspension of the standard microorganism, prepared
purity of prepared compounds were examined by from 24 h cultures of bacteria in blood agar and from 24 h
TLC plates technique (DC-Alufolien Kiesegel 60 F256
Merck).
,
cultures in the Sabouraud agar for fungi had a
concentration of 5×107 cfu mL-1 of bacteria and 5×105 cfu mL-1
of Candida. The suspensions were prepared in
physiological solution (pH 7.2). Concentration of
microorganismsweredeterminedspectrophotometrically
and the suspensions were adjusted to absorbanceA= 0.35 at
λ = 540 nm. The microorganism suspension (5 µL)
was added to solutions containing the compound tested
(100 µL) and to double concentrated peptone broth
medium (8%) for bacteria or Sabouraud medium (12%)
for Candida (100 µL). The stock solutions of the
compounds tested had a concentration of 1000 µg mL-1.
For a testing assay the solutions were serially diluted by
half (concentrations of the solutions were: 1000, 500,
250, 125, 62.5, 31.25, 15.63, 7.81, 3.91 and 1.95 µg mL-1)
and used. The cultures were done in 96-well microtiter
plates. The microorganisms were incubated for 24 h at
37°C. Then from each well, 5 µL of suspension tested
was taken and cultured on blood agar (bacteria) or on
Sabouraud agar (fungi). The Petri dishes were incubated
for 24 h at 37°C. The lowest concentration of QUATs
that prevented colony formation was defined as the
MBC.
2.2. Synthesis of (2S)-N-alkyl-N,N-dimethyl-
(1-hydroxy-3-phenylpropyl)-2-ammonium
bromide and (2R)-N-alkyl-N,N-dimethyl-
(1-hydroxy-3-phenylpropyl)-2-ammonium
bromide
(2S)-2-Amino-3-phenyl-1-propanol1and(2R)-2-Amino-
3-phenyl-1-propanol 2 were prepared as previously
described [40].
(2S)-N,N-dimethyl-2-amino-3-phenylpropan-1-ol
3
and (2R)-N,N-dimethyl-2-amino-3-phenylpropan-1-ol 4
were prepared by a previously reported method [34].
(2S)-N-decyl-N,N-dimethyl-(1-hydroxy-3-
phenylpropyl)-2-ammonium bromide 5S. (2S)-N,N-
Dimethyl-2-amino-3-phenyl-1-propanol(5.74g,32.0mmol)
and 1-bromodecane (8.36 g, 32.0 mmol) were added to
25 mL of acetonitrile, then heated under reflux for 16 h.
The reaction mixture was cooled to room temperature
and evaporated. Diethyl ether was added to precipitate
the ammonium salt. The precipitate was filtered off and
the crude product was recrystallized repeatedly from
acetone-petrolether mixture.
(2S)-N-dodecyl-N,N-dimethyl-(1-hydroxy-3-
phenylpropyl)-2-ammoniumbromide6S, (2S)-N-tetradecyl-
N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium
bromide 7S, (2S)-N-hexadecyl-N,N-dimethyl-(1-hydroxy-3-
phenylpropyl)-2-ammonium bromide 8S, (2S)-N-octadecyl-
N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium
bromide 9S, (2R)-N-decyl-N,N-dimethyl-(1-hydroxy-3-
phenylpropyl)-2-ammonium bromide 5R, (2R)-N-dodecyl-
N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium
bromide 6R, (2R)-N-tetradecyl-N,N-dimethyl-(1-hydroxy-
3-phenylpropyl)-2-ammonium bromide 7R, (2R)-N-
hexadecyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-
2-ammonium bromide 8R were prepared by the same
procedure as 5S, by alkylation of tertiary amine with the
corresponding 1-bromoalkane.
2.4. QSAR study of (2S)-N-alkyl-N,N-dimethyl-
(1-hydroxy-3-phenylpropyl)-2-ammonium
bromides
The relationhips were calculated using Kubinyi’s
bilinear model. QSAR was performed on a series of
compounds 5S – 9S. Programe BILIN which is available
at http://www.kubinyi.de/bilin-program.html was used
for computation.
3. Results and discussion
QUATs derived from phenylalanine 5S-9S and 5R-8R
(Table 1) were synthesized according to the procedure
shown in Fig. 1. Five of the compounds were prepared as
S-derivatives (5S-9S) and four as R-derivatives (5R-8R),
in which the alkyl chains range from C10 (compound 5)
to C18 (compound 9). The derivatives with different alkyl
2.3. Bactericidal and fungicidal studies
The microbicidal activity was tested against Gram- chain were prepared by reaction of the tertiary amine
negativebacteriaEscherichiacoliCNCTC377/79,Gram- with the appropriate 1-bromoalkane. Quaternisation of
196

M. Lukáč et al.
COOH
NH
OH
(ii)
(i)
(iii)
OH
OH
+
NH
-
2
N
N
2
Br
(CH )n
2
Figure 1. Synthesis of QUATs derived from phenylalanine. Reagents and conditions: (i) Na[BH4], I2, THF, reflux, 70 – 94%; (ii) 35% HCHO, 85%
HCOOH, reflux, 86 – 90%; 1-bromoalkane, CH3CN, reflux, 57 – 86%
agreement with the expected structures and showed
essentially no contaminants (Table 2). Extensive NMR
spectral data, including 2D COSY experiment, provided
strong support for the structure of the product 6R and
analogically for other QUATs. Purity of substances was
also confirmed by TLC. IR spectra showed characteristic
vibrations of the following groups of atoms: ν(O-H),
ν(C-O), ν(C-H, Ph), ν(C=C). Abundance of atoms of C,
H, N was specified with elemental analysis.
Consequently, the microbicidal activities of the
target molecules 5S-9S and 5R-8R and their equimolar
mixtures rac-5–rac-8 were measured with Escherichia
coliCNCTC377/79asarepresentativeofGram-negative
bacteria; Staphylococcus aureus ATCC 6538 as a
phenylalaninols was carried out with 1-bromodecane,
1-bromododecane, 1-bromotetradecane and
1-bromohexadecane; in the case of both enantiomers of
N,N-dimethyl-2-amino-3-phenylpropan-1-ols. (2S)-N,N-
dimethyl-2-amino-3-phenylpropan-1-ol was quaternised
also with 1-bromooctadecane. The substances were
obtained as white crystals after several crystallizations
from acetone-methanol or acetone-petroleum ether.
The yields ranged from 57 to 87%. Melting points were
well defined and their values slightly increased based
on dependence of the growing alkyl chains. The value
of optical rotation also depended on the length of the
alkyl chain. Optical rotation declined with growing alkyl
1
chain (Table 1). H and 13C NMR spectra were in good
Table 1. Yields and physicochemical properties of prepared compounds
[α]2D5
Compound
Number
of atom
in the
alkyl
chain
Formula
Mr
wi(calc.)/%
Yield
M.p.a
TLCb
wi(found)/%
H
C
N
%
°C
Rf
5S
6S
7S
8S
9S
5R
6R
7R
8R
10
12
14
16
18
10
12
14
16
C21H38BrNO
400.44
62.99
63.13
9.57
9.84
3.50
3.60
56.8
112.5 – 114
-6.19
-5.96
-5.69
-5.36
-5.02
6.08
6.01
5.82
5.13
0.47
C23H42BrNO
428.49
64.47
64.21
9.88
10.07
3.27
3.33
85.6
68.0
73.5
72.3
63.9
76.8
65.2
75.5
114.5 – 115.5
116 – 117.5
117 – 118.5
119 – 120
0.49
0.47
0.49
0.51
0.47
0.48
0.49
0.51
C25H46BrNO
456.54
65.77
64.74
10.15
10.23
3.07
3.09
C27H50BrNO
484.60
66.92
66.79
10.40
10.70
2.89
2.94
C29H54BrNO
512.65
67.94
67.82
10.62
10.89
2.73
2.78
C21H38BrNO
400.44
62.99
63.18
9.57
9.84
3.50
3.56
113 – 114
C23H42BrNO
428.49
64.47
64.76
9.88
10.14
3.27
3.37
114.5 – 115
116 – 117,5
118 – 119
C25H46BrNO
456.54
65.77
66.45
10.15
10.54
3.07
3.17
C27H50BrNO
484.60
66.92
67.25
10.40
10.78
2.89
2.96
a Solvent for recrystallization CH3COCH3-petrolether (5S, 5R), CH3COCH3-MeOH (6S-9S, 6R-8R)
b CHCl3 : MeOH – 5 : 1 (V/V)
197
197

Synthesis and antimicrobial activity of a series of optically active
quaternary ammonium salts derived from phenylalanine
negative bacteria Escherichia coli and Gram-positive
bacteria Staphylococcus aureus were exhibited by
compounds 7S, 7R and rac-7 with alkyl chain length
n=14. The remaining compounds with either longer or
shorter alkyl chains exhibited lower activity compared
to compounds 7S, 7R and rac-7. For the S. aures,
the cut-off effect was observed at 14.7, i.e.,15 carbon
atoms in the chain (Table 4). For E.coli this parameter
is 13.5, i.e.,14 carbon atoms in the chain. Similarly for
C. albicans the Noptimal is 14.6, i.e.,15 carbon atoms in
the chain. The compounds 8S, 8R and rac-8 exhibited
the highest activity against Candida albicans with their
representative of Gram-positive bacteria; and Candida
albicans CCM 8186 as a representative of fungi. The
minimal bactericidal or fungicidal concentration (MBC)
values were defined as the minimal concentration of the
antimicrobial agent that killed the test organism after
24 h of incubation. The MBC values determined for the
compounds prepared are listed in Table 3.
Microbicidal activities of the prepared compounds
were determined as a function of the length of the alkyl
chain, which ranged from 10 – 18 for (2S)-N-alkyl-N,N-
dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium
bromides and 10 – 16 for (2R)-N-alkyl-N,N-dimethyl-
alkyl chain lengths being 16 carbon atoms. Comparable
activity was found in compounds 7S, 7R and rac-7 also.
The MBC values of compounds 7S and 7R and their
racemate taken against Candida albicans were approx.
0.5 µM lower than the MBC values of compounds 8S,
8R and rac-8 (Table 3). We also observed that the
(1-hydroxy-3-phenylpropyl)-2-ammonium
bromides,
respectively (Table 3). Fig. 2 shows the relative
bactericidal and fungicidal activities (log1/MBC) of (2S)-
N-alkyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-
ammonium bromides based on the length of the alkyl
chain. The highest bactericidal activity against Gram-
Table 2. Spectral data of prepared compounds
Compound
Spectral data
-1
~
5S
IR: /cm : 1032 (C-O), 1605 (C=C), 2923 (C-H, alkyl chain), 3025 (C-H, Ph), 3295 (O-H)
v
n
1H NMR (CDCl3), δ: 0.88 (t, J = 6.7 Hz, 3H, CH2CH3), 1.26 (m, 14H, alkyl chain), 1.72 (2H, m NCH2CH2), 3.12 (m, 1H, 3-HH),
3.31 (m, 1H, 3-HH), 3.38 (s, 3H, N(CH3)(CH3)), 3.45 (s, 3H, N(CH3)(CH3)), 3.55 (m, 1H, N-CHHCH2), 3.61 (m, 1H, 2-H) 3.71
(m, 1H, CHHOH), 3.82 (m, 1H, N-CHHCH2), 4.21 (m, 1H, CHHOH) 5.09 (t, J = 5.0 Hz, 1H, CHHOH), 7.2 – 7.4 (m, 5H, Ph)
13C NMR (CDCl3), δ: 14.13 (CH3), 22.68, 22.92, 26.31, 29.26, 29.37, 29.42, 31.06, 31.83 (8 × CH2), 50.43 (2 × N-CH3), 56.36
(N-CH2), 64.75 (C-2), 73.82 (C-1), 127.52 (C-4’), 129.13 (2 × C), 129.43 (2 × C), 135.48 (C-1’)
~
6S
7S
8S
9S
IR: v /cm-1: 1032 (C-O), 1605 (C=C), 2921 (C-H, alkyl chain), 3024 (C-H, Ph), 3296 (O-H)
n
1H NMR (CDCl3), δ: 0.88 (t, 3H, J = 6.7 Hz, CH2CH3), 1.25 (m, 18H, alkyl chain), 1.74 (2H, m NCH2CH2), 3.12 (m, 1H, 3-HH),
3.33 (m, 1H, 3-HH), 3.38 (s, 3H, N(CH3)(CH3)), 3.44 (s, 3H, N(CH3)(CH3)), 3.52 (m, 1H, N-CHHCH2), 3.60 (m, 1H, 2-H) 3.71
(m, 1H, CHHOH), 3.82 (m, 1H, N-CHHCH2), 4.21 (m, 1H, CHHOH) 5.08 (t, J = 5.0 Hz, 1H, CHHOH), 7.2 – 7.4 (m, 5H, Ph)
13C NMR (CDCl3), δ: 14.12 (CH3), 22.66, 22.93, 26.31, 29.25, 29.33 29.37, 29.42, 29.59, 31.06, 31.83 (10 × CH2), 50.43 (2 ×
N-CH3), 56.38 (N-CH2), 64.74 (C-2), 73.82 (C-1), 127.51 (C-4’), 129.12 (2 × C), 129.44 (2 × C), 135.52 (C-1’)
~
IR: /cm-1: 1032 (C-O), 1604 (C=C), 2923 (C-H, alkyl chain), 3024 (C-H, Ph), 3294 (O-H)
v
n
1H NMR (CDCl3), δ: 0.88 (t, J = 6.7 Hz, 3H, CH2CH3), 1.26 (m, 22H, alkyl chain), 1.73 (2H, m NCH2CH2), 3.12 (m, 1H, 3-HH),
3.29 (m, 1H, 3-HH), 3.38 (s, 3H, N(CH3)(CH3)), 3.45 (s, 3H, N(CH3)(CH3)), 3.55 (m, 1H, N-CHHCH2), 3.59 (m, 1H, 2-H) 3.71
(m, 1H, CHHOH), 3.82 (m, 1H, N-CHHCH2), 4.21 (m, 1H, CHHOH) 5.09 (t, J = 5.1 Hz, 1H, CHHOH), 7.2 – 7.4 (m, 5H, Ph)
13C NMR (CDCl3), δ: 14.12 (CH3), 22.69, 22.92, 26.31, 29.26, 29.36, 29.48, 29.60, 29.64, 29.68, 31.06, 31.92 (12 × CH2),
50.43 (2 × N-CH3), 56.36 (N-CH2), 64.78 (C-2), 73.86 (C-1), 127.50 (C-4’), 129.14 (2 × C), 129.42 (2 × C), 135.49 (C-1’)
IR: /cm-1: 1032 (C-O), 1605 (C=C), 2920 (C-H, alkyl chain), 3024 (C-H, Ph), 3300 (O-H)
~
v
n
1H NMR (CDCl3), δ: 0.88 (t, J = 6.7 Hz, 3H, CH2CH3), 1.26 (m, 26H, alkyl chain), 1.73 (2H, m NCH2CH2), 3.13 (m, 1H, 3-HH),
3.32 (m, 1H, 3-HH), 3.39 (s, 3H, N(CH3)(CH3)), 3.47 (s, 3H, N(CH3)(CH3)), 3.55 (m, 1H, N-CHHCH2), 3.60 (m, 1H, 2-H) 3.71
(m, 1H, CHHOH), 3.81 (m, 1H, N-CHHCH2), 4.20 (m, 1H, CHHOH) 5.08 (t, J = 5.0 Hz, 1H, CHHOH), 7.2 – 7.4 (m, 5H, Ph)
13C NMR (CDCl3), δ: 14.14 (CH3), 22.69, 22.92, 26.32, 29.27, 29.37, 29.49, 29.61, 29.66, 29.70, 31.06, 31.92 (14 × CH2),
50.42 (2 × N-CH3), 56.38 (N-CH2), 64.74 (C-2), 73.81 (C-1), 127.50 (C-4’), 129.12 (2 × C), 129.46 (2 × C), 135.52 (C-1’)
~
-1
v
IR: /cm : 1032 (C-O), 1605 (C=C), 2923 (C-H, alkyl chain), 3025 (C-H, Ph), 3298 (O-H)
n
1H NMR (CDCl3), δ: 0.88 (t, J = 6.7 Hz, 3H, CH2CH3), 1.26 (m, 30H, alkyl chain), 1.73 (2H, m NCH2CH2), 3.12 (m, 1H, 3-HH),
3.30 (m, 1H, 3-HH), 3.38 (s, 3H, N(CH3)(CH3)), 3.44 (s, 3H, N(CH3)(CH3)), 3.55 (m, 1H, N-CHHCH2), 3.60 (m, 1H, 2-H) 3.71
(m, 1H, CHHOH), 3.82 (m, 1H, N-CHHCH2), 4.20 (m, 1H, CHHOH) 5.09 (t, J = 5.1 Hz, 1H, CHHOH), 7.2 – 7.4 (m, 5H, Ph)
13C NMR (CDCl3), δ: 14.12 (CH3), 22.66, 22.93, 26.31, 29.25, 29.37, 29.42, 31.06, 31.83 (16 × CH2), 50.43 (2 × N-CH3),
56.38 (N-CH2), 64.74 (C-2), 73.82 (C-1), 127.50 (C-4’), 129.12 (2 × C), 129.45 (2 × C), 135.52 (C-1’)
198

M. Lukáč et al.
antimicrobial activities of QUATs depend strongly on observed only in one dilution. The microbicidal activity of
the structure of their alkyl chain. If they possessed an racemates (equimolar mixtures of pure enatiomers) was
unbranched alkyl chain and the head group did not the same as the MBC values of individual enantiomers.
contain a hydrophilic or lipophilic moiety, the optimal Similar results were published by Osanai and Abe [56]
alkyl chain length is usually 14-16 carbon atoms [42- with QUATs derived from 1-phenylethanamine.
48]. N-alkyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-
We compared the microbicidal activity of N-alkyl-
2-ammonium bromides fulfills these conditions. The N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium
alkyl chain is straight, unbranched, bearing no aromatic bromides with the activity of commercially available
rings or heteroatoms. The head group contains one benzalkonium bromide (BAB). MBC values of QUATs
lipophilic moiety – a benzyl group and one hydrophilic 7S, 8S, 7R, 8R, rac-7, rac-8 against Gram-positive
moiety – a hydroxyl group and their hydrophilic-lipophilic bacteria Staphylococcus aureus were found to be lower
balance can be compensated. Compounds 7S, 7R and than that of BAB. The MBC of compound 9S was higher
rac-7 contain a C14 alkyl chain and so one can expect against Escherichia coli than the MBC of BAB. All the
the highest microbicidal activity in the series of QUATs other compounds have lower MBCs than BAB. The
investigated with these compounds.
fungicidal activity of QUATs with alkyl chain length n =
We also studied the quantitative relationship between 12 – 16 against Candida albicans were observed to be
structure and activity (QSAR) using the equation higher than that of BAB. Compounds 7S, 8S, 7R, 8R,
log (1/MBC) = f(n), where n is the number of carbon
atoms in the long alkyl chain. This non-linear course was
quantified by the Kubinyi’s bilinear model [49,50]
Table 3. The MBC valuesa of prepared QUATs
Compound
S. aureus
ATCC
6538
E. coli
CNCTC
377/79
C.albicans
CCM 8186
log (1/MBC) = An + Blog (β 10n + 1) + C
5S
6S
195.1
36.4
8.5
195.1
18.1
8.5
390.2
18.1
8.5
This Kubinyi´s bilinear model was also used by
evaluation of rich series of compounds described in
our earlier papers [42,43,51-55] and its predictive
power was proved many times. The values for
compounds described in this paper are presented
in Table 4. From the computations it follows (Table
4) that the highest activity of this type of compounds
would be with the alkyl chain length C15 for S. aureaus
(MBC = 7.6 µM) and C. albicans (MBC = 4.6 µM),
and C14 for E. coli (MBC = 8.5 µM), respectively. The
values given in parentheses are calculated values from
the bilinear equation.
We have also studied the relationship between
the optical activity of these compounds and their
microbicidal activity. We observed that the optical activity
of enantiomers of N-alkyl-N,N-dimethyl-(1-hydroxy-3-
phenylpropyl)-2-ammonium bromides had no effect
on their bactericidal or fungicidal activity. Differences
in microbicidal activity between S and R enantiomers
of QUATs with same length of the alkyl chain were
7S
8S
16.1
121.9
195.1
18.2
8.5
64.4
975.3
195.1
18.1
8,5
8.0
9S
61.0
390.2
18.1
8.5
5R
6R
7R
8R
16.1
195.1
36.4
8.5
64.4
195.1
18.1
8.5
8.0
rac-5
rac-6
rac-7
rac-8
BAB
390.2
18.1
8.5
16.1
26.0
64.4
260.1
8.0
26.0
Table 4. Regression coefficients for bilinear relationship between microbicidal activity and structure for (2S)-N-alkyl-N,N-dimethyl-(1-hydroxy-3-
phenylpropyl)-2-ammonium bromides
b
d
Relationship
S. aureus
A
B
C
logβ
-14.70
-13.51
-14.59
ra
sD
Fc
Noptimal
14.6
MBCoptimal
0.357±0.011
0.512±0.077
0.453±0.306
-0.803±0.025
-1.063±0.125
-0.859±0.649
0.148±0.136
-1.402±0.904
-0.987±3.736
1.000
0.999
0.963
0.014
0.074
0.383
2158.634
162.697
4.229
7.0
6.4
4.3
E. coli
13.5
C. albicans
14.6
199
199

Synthesis and antimicrobial activity of a series of optically active
quaternary ammonium salts derived from phenylalanine
5,4
5,2
5,0
4,8
4,6
4,4
4,2
4,0
3,8
3,6
3,4
3,2
3,0
2,8
10
12
14
16
18
n
Figure 2. Relation between alkyl chain length and microbicidal activity of (2S)-N-alkyl-N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammo-
C. albicans.
E. coli
S. aureus
niumbromides; Symbols:
rac-7, rac-8 exhibit very good microbicidal activity, and
in comparison to BAB, they are more effective against
all the microorganisms studied.
of phenylalanine−derived QUATs have apparently no
influence on their microbicidal activity. The relationship
between MBC and alkyl chain length of (2S)-N-alkyl-
N,N-dimethyl-(1-hydroxy-3-phenylpropyl)-2-ammonium
bromides was quantified by a bilinear model. The
results show that Staphylococcus aureus would be
most sensitive towards the compound with a C15 chain,
Escherichia coli towards C14 and Candida albicans
towards C15, respectively.
4. Conclusions
The structure−activity relationship of QUATs derived
from phenylalanine was investigated to determine the
influence of alkyl chain length and stereochemistry of
surfactants on their microbicidal activities. The cut-off
effect which was observed, wherein the antimicrobial
activity depended on the length of alkyl chain, was
quantified by a bilinear model. Compounds No. 7S, 7R
and rac-7 exhibited the highest microbicidal activity
from among the compounds studied. The results
suggest that stereochemistry in terms of optical activity
Acknowledgements
This work was supported by the grants Nos.:
UK/110/2008, VEGA 1/4300/07, VEGA 1/3416/06,
VEGA1/4290/07 and VEGA 1/0164/08.
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