molecules

Article
Synthesis, Structure and Antimicrobial Properties of
Novel Benzalkonium Chloride Analogues with
Pyridine Rings
Bogumił Brycki 1, *, Izabela Małecka 1 , Anna Koziróg 2 and Anna Otlewska 2
1 Laboratory of Microbiocides Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Poznan,
Umultowska 89b, 61-614 Poznań, Poland; imalecka@amu.edu.pl
2 Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences,
Lodz University of Technology, Wólczańska 171/173, 90-924 Łódź, Poland; anna.kozirog@p.lodz.pl (A.K.);
anna.otlewska@p.lodz.pl (A.O.)
* Correspondence: brycki@amu.edu.pl; Tel.: +48-61-829-1694

Academic Editor: Derek J. McPhee

Received: 9 December 2016; Accepted: 9 January 2017; Published: 13 January 2017

Abstract: Quaternary ammonium compounds (QACs) are a group of compounds of great economic
significance. They are widely used as emulsifiers, detergents, solubilizers and corrosion inhibitors
in household and industrial products. Due to their excellent antimicrobial activity QACs have also
gained a special meaning as antimicrobials in hospitals, agriculture and the food industry. The main
representatives of the microbiocidal QACs are the benzalkonium chlorides (BACs), which exhibit
biocidal activity against most bacteria, fungi, algae and some viruses. However, the misuses of
QACs, mainly at sublethal concentrations, can lead to an increasing resistance of microorganisms.
One of the ways to avoid this serious problem is the introduction and use of new biocides with
modified structures instead of the biocides applied so far. Therefore new BAC analogues P13–P18
with pyridine rings were synthesized. The new compounds were characterized by NMR, FT-IR and
ESI-MS methods. PM3 semiempirical calculations of molecular structures and the heats of formation
of compounds P13–P18 were also performed. Critical micellization concentrations (CMCs) were
determined to characterize the aggregation behavior of the new BAC analogues. The antimicrobial
properties of novel QACs were examined by determining their minimal inhibitory concentration
(MIC) values against the fungi Aspergillus niger, Candida albicans, Penicillium chrysogenum and bacteria
Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The MIC values of
N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides for fungi range from 0.1 to 12 mM
and for bacteria, they range from 0.02 to 6 mM.

Keywords: quaternary ammonium salts; benzalkonium chlorides; biocides; antimicrobial resistance;

minimal inhibitory concentration; critical micellization concentrations

1. Introduction
Quaternary ammonium compounds (QACs) are a group of cationic surfactants widely used in
household, agricultural, clinical and industrial products. QACs are surface active organic compounds
containing at least one long hydrocarbon substituent attached covalently to a positively charged
quaternary nitrogen atom. The other substituents can be alkyl, aromatic, ether or ester groups.
Owing to the varied structures and a wide span of hydrophilic–lipophilic balance QACs are extensively
used as emulsifiers [1], dispersants [2,3], fabric softeners [4], cosmetic ingredients [5] and phase transfer
catalysts [6,7], gene delivery agents [8], antimicrobial [9,10] and anticorrosion [11,12] agents.
Among a large number of cationic surfactants, examples of which are shown in Figure 1,
benzalkonium chlorides 3 deserve the special attention due to their unique biocidal properties.

Molecules 2017, 22, 130; doi:10.3390/molecules22010130 www.mdpi.com/journal/molecules

Molecules 2017, 22, 130 2 of 12

Molecules 2017, 22, 130 2 of 12

They were presented for the first time as antimicrobial agents by Domagk in 1935 [13]. Nowadays,
werewidely
they are presented used foras
thedisinfectants
first time as antimicrobial agentsas
and antiseptics, bywell
Domagk in 1935 [13]. Nowadays,
as preservatives they arenasal
for ophthalmic,
widely used as disinfectants and antiseptics, as well as preservatives for ophthalmic, nasal and
and inhalational drugs.
Molecules 2017, 22, 130 They are also active substances in medicines for throat and mouth 2infections. of 12
inhalational drugs. They are also active substances in medicines for throat and mouth infections. The
The mechanism of their biocidal action toward most bacteria, fungi and algae is based on adsorption
mechanism of their biocidal action toward mostagents
bacteria,
by fungi andinalgae is based on adsorption of
of thewere
the
presented
alkylammonium
alkylammonium
for the first time
cation
cation on
as
the
onand
antimicrobial
bacterialcell
theantiseptics,
bacterial cellsurface, Domagk
surface, diffusion 1935 [13].
through Nowadays,
the the
cell cell
they are
wall
and and then
widely used as disinfectants as well as diffusion through
preservatives for ophthalmic,wallnasal then
and
binding and and
binding disruption
disruption of cytoplasmatic membrane. Damagetotothethe membrane results
in a in a release
of of
inhalational drugs. Theyof cytoplasmatic
are membrane.
also active substances in Damage
medicines membrane
for throat results
and mouth release
infections. The
potassium ions and other cytoplasmatic constituents, finally leading to the death of
potassium ions and other cytoplasmatic constituents, finally leading to the death of the cell [14–17]. the cell [14–17].
mechanism of their biocidal action toward most bacteria, fungi and algae is based on adsorption of
the alkylammonium cation on the bacterial cell surface, diffusion through the cell wall and then
binding and disruption of cytoplasmatic membrane. Damage to the membrane results in a release of
potassium ions and other cytoplasmatic constituents, finally leading to the death of the cell [14–17].

FigureFigure
1. 1. Examplesofofantimicrobial
Examples antimicrobial quaternary
quaternary ammonium
ammoniumhalides:
halides:alkylpyridinium
alkylpyridinium(1); (1);
alkyltrimethylammonium (2); benzalkonium (3); dialkyldimethylammonium (4) and diesteralkonium
alkyltrimethylammonium (2); benzalkonium (3); dialkyldimethylammonium (4) and diesteralkonium (5). (5).

However, in recent years there have been frequent reports of cases of increasing resistance of
Figure 1. Examples of antimicrobial quaternary ammonium halides: alkylpyridinium (1);
However, in recent years
certain microorganism there
strains to the have beenactivity
biocidal frequent reports of cases
of benzalkonium of increasing
chlorides [18–21]. The resistance
use of of
alkyltrimethylammonium (2); benzalkonium (3); dialkyldimethylammonium (4) and diesteralkonium (5).
certain microorganism
inappropriate strains to
concentrations the biocidal activity
of benzalkonium chloridesof andbenzalkonium
incomplete removal chlorides [18–21].
of residues The use
of these
microbiocides
of inappropriate after finishing
concentrations the
of disinfection
benzalkonium process exposes
chlorides microorganisms
and incomplete
However, in recent years there have been frequent reports of cases of increasing resistance of to prolonged
removal of contact
residues of
with
thesecertain sublethal
microbiocides doses
afterof BACs.
finishingThis contributes
the disinfectionto the development
process exposesof defensive
microorganism strains to the biocidal activity of benzalkonium chlorides [18–21]. The use of mechanisms
microorganisms to and the
prolonged
contactemergence
inappropriate of concentrations
with sublethal strains of microorganisms
doses of of
BACs. Thisexhibiting
benzalkonium contributes
chlorides resistance
to
andthe to benzalkonium
development
incomplete removal of chlorides
defensive
of residues asmechanisms
ofwell
theseas
cross-resistance
microbiocides to
after certain
finishingother antibiotics [22,23]. This fact creates a serious epidemiological threat,
and the emergence of strains ofthe disinfection process
microorganisms exposesresistance
exhibiting microorganisms to prolonged chlorides
to benzalkonium contact as
especially in medical facilities, manifested by antoincrease in morbidity and mortality among and patients
well with sublethal
as cross-resistance doses to of BACs.
certainThis
othercontributes
antibiotics the development
[22,23]. This fact of creates
defensive mechanisms
a serious the
epidemiological
[24]. One ofofthe
emergence way to ofovercome this serious negative side effect is a periodicallychlorides
application of new
threat, especially strains
microbiocides in medical
with
microorganisms
modified facilities,
structures.
exhibiting
manifested byresistance
an increase to benzalkonium
in morbidity and mortality as well as
among
cross-resistance to certain other antibiotics [22,23]. This fact creates a serious epidemiological threat,
patients [24]. One
To meet of the way
this challenge to overcome
wemanifested
have proposedthis serious negative side effect is a periodically application
especially in medical facilities, by an aincrease
modification of benzalkonium
in morbidity and mortality chlorides
amongbasedpatients on
of new themicrobiocides
introduction of with
nitrogenmodified
atoms structures.
into the aromatic ring. For that reason a series of novel quaternary
[24]. One of the way to overcome this serious negative side effect is a periodically application of new
To meet thiscompounds
ammonium
microbiocides challenge
with modified we have
P13–P18 proposed
with
structures. variable a modification
alkyl chain length of benzalkonium
and bearing pyridine chloridesringsbased
instead on the
of phenyl
introduction ofrings were
nitrogen synthesized.
atoms into In
the the literature
aromatic there
ring. are
For known
that nonionic
reason
To meet this challenge we have proposed a modification of benzalkonium chlorides based on a surfactants
series of novelcontaining
quaternary
4-pyridyl
ammonium
the moieties,
compounds
introduction for example
P13–P18
of nitrogen atoms amines
with [25–27],
intovariable
the amides
alkyl
aromatic chain
ring. [28],
For ethers
length
that and
reasonandbearing
a ketones [29,30],
series ofpyridine asrings
well instead
novel quaternary as
4-alkylpyridines
of phenyl
ammonium rings compounds[31]. According
were synthesized. P13–P18 In to our
with knowledge
thevariable
literature this
alkylthere is the
chainare first
known
length report however
nonionic
and bearing about the
surfactants
pyridine synthesis
containing
rings instead
of phenyl
of a seriesrings
of benzalkonium chloride In analogues bearing
there4-pyridyl rings.
4-pyridyl moieties,were synthesized.
for example amines the[25–27],
literature amides are known
[28], ethersnonionic surfactants
and ketones containing
[29,30], as well as
4-pyridyl
4-alkylpyridines moieties,
[31]. for example amines [25–27], amides [28], ethers and
According to our knowledge this is the first report however about the synthesisketones [29,30], as well as
2. Results and Discussion
4-alkylpyridines [31]. According to our knowledge this is the first report however about the synthesis
of a series of benzalkonium chloride analogues bearing 4-pyridyl rings.
of a series of benzalkonium
The novel chloride analogues
compounds P13–P18 bearing
were obtained 4-pyridyl
according rings. 1 by Menshutkin reaction of
to Scheme
4-chloromethylpyridine
2. Results and Discussion (6) with the corresponding N,N-dimethylalkylamines 7–12 containing 8, 10,
2.
12,Results and
14, 16, 18 Discussion
carbon atoms in alkyl chain, respectively. The use of the polar aprotic solvent acetonitrile
The novel compounds
as a reaction medium P13–P18
allowed us towere obtained
obtain according
compounds P13–P18 toinScheme 1 byatMenshutkin
good1yield reaction of
room temperature.
The novel compounds P13–P18 were obtained according to Scheme by Menshutkin reaction of
4-chloromethylpyridine
4-chloromethylpyridine (6)(6)
with
withthe
thecorresponding N,N-dimethylalkylamines
corresponding N,N-dimethylalkylamines 7–127–12 containing
containing 8, 10, 8, 10,
12, 14, 7, P13 n=5
12,16,
14,18
16,carbon atoms
18 carbon atoms inin
alkyl
alkylchain,
chain,respectively. Theuse
respectively. The useofof
thethe polar
polar
e
aprotic
aprotic solvent
solvent acetonitrile
acetonitrile
N CH 3CN N 8, P14 n=7
as a reaction medium
as a reaction mediumallowed
+ allowed us
N to
us obtain
to obtain compounds
compounds P13–P18
P13–P18 inin good
good yield
yield at at
roomroom temperature.
temperature.
9, P15 n=9
Cl r.t. a c N Cl i
n g 10, P16 n=11
b d f hn 7, P13
11, P17 n=5
n=13
N 6 7-12 N eP13-P18 8, P14
12, P18 n=15
n=7
CH 3CN
+ N 9, P15 n=9
Cl r.t. a c N Cl i
n Synthesis
Scheme 1. of compounds P13–P18. g 10, P16 n=11
b d f hn 11, P17 n=13
6 7-12 P13-P18 12, P18 n=15

Scheme 1. Synthesis of compounds P13–P18.

Scheme 1. Synthesis of compounds P13–P18.
Molecules 2017, 22, 130 3 of 12

Crude products were purified by crystallization from 1:1 acetonitrile/acetone mixtures and
characterized by spectroscopic methods. Copies of recorded spectra of compounds P13–P18 are
available in Supplementary Materials.

2.1. Spectroscopic Characterization

2.1.1. Nuclear Magnetic Resonance Spectra

The most characteristic feature of the 1 H-NMR spectra of the title compounds are the signals
of the pyridine protons and the protons of the methyl and methylene groups directly linked to the
positively charged nitrogen atom. The chemical shifts of the H-a and H-b protons of the pyridine ring
are observed around 8.70–8.71 and 7.72–7.73 ppm, respectively. They are shifted toward the higher
ppm values than those observed for γ-picoline [32], for example. This is caused by the proximity
of the positively charged nitrogen atom linked via methylene groups to the C-c carbon atom of the
pyridine ring (Scheme 1). Protons H-d and H-e of then methylene and methyl groups directly attached
to the quaternary nitrogen atom appear in 1 H-NMR spectra as singlets in the 5.30–5.24 ppm and
3.54–3.52 ppm regions, respectively. In all 1 H-NMR spectra of the analyzed surfactants P13–P18 H2 O
proton signals are observed in the range 2.98–2.65 ppm. According to literature data the protons of
H2 O in CDCl3 resonate at 1.56 ppm [33]. The observed shifts over 1 ppm toward the higher ppm
values in compounds P13–P18 spectra indicate the presence of weak hydrogen bonding between the
water molecules and chloride anions.
The 13 C-NMR spectra of P13–P18 show three characteristic groups of signals in the range of
150.65–150.70, 127.58–127.59 and 136.11–136.15 ppm, which are assigned to C-a, C-b and C-c carbon
atoms of the pyridine rings, respectively. The carbon atoms of the methylene groups C-d and C-f
resonate at 65.58–65.49 ppm and 64.07–64.02 ppm, respectively, whereas the carbon atoms of the
methyl group C-e are observed at 49.88–49.79 ppm.

2.1.2. Infrared Spectra

In the FT-IR spectra of P13–P18 some a very characteristic bands are observed. The stretching
vibrations bands of pyridine C–H bonds are observed in the range 3062–3005 cm−1 while deformational
vibrations, in plane and out of plane, lie in the 1351–1069 cm−1 and 829–820 cm−1 regions, respectively.
Stretching vibrations bands of aromatic ring C=C and C=N bonds are observed in a typical
1617–1562 cm−1 region. Deformational and skeletal vibrations bands of the pyridine rings are observed
in the 1419–1415 cm−1 and 1006–996 cm−1 regions, respectively. The symmetric and asymmetric
stretching vibrations of C–H bonds of methyl and methylene groups lie in the 2985–2850 cm−1 region.
Bands observed in the 3454–3242 cm−1 region are assigned to stretching vibrations of hydrogen bonded
H2 O molecules.

2.1.3. ESI-MS
In ESI-MS in positive ion mode of all analyzed surfactants the presence of the corresponding [M+ ]
ion was observed as the strongest peak. The presence of a [2M + Cl]+ ion peak was also observed.
For all discussed compounds in negative ion mode the presence of a [M + 2Cl]− ion was observed.
Taken together, the ESI-MS spectra, as well as 1 H-NMR, 13 C-NMR and FT-IR spectra confirmed the
structure and purity of the obtained compounds.

2.2. Semiempirical Calculations

PM3 semiempirical calculations were performed using the Gaussian03W program.
Some representative molecular models for P13, P15 and P17 are shown in Figure 2. The heats
of formation (HOF) and geometric parameters of compounds P13–P18 are presented in Table 1.
The spatial arrangement of atoms around the tetrahedral quaternary nitrogen atom as well as the
geometry of the alkyl chains of compounds P13–P18 are very similar to those of other alkyl ammonium
Molecules 2017, 22, 130 4 of 12

salts [34]. The change from phenyl ring to pyridine ring has a little effect on the geometric parameters.
Similarly the nature of the anion, bromide or chloride, has practically no effect on the bond lengths and
angles in alkylammonium salts. What is more, the calculated parameters in gas phase correspond to
some extent to the parameters of the molecule geometry in crystal form (Table 1).

Table 1. HOF, bond lengths and angles for P13–P18, BDDAC and BDDAB.
Molecules 2017, 22, 130 4 of 12
Compound
What is more, the calculated parameters in gas phase correspond to some extent1 to theBDDAB
BDDAC 2
parameters ofBDDAB 3
P13 P14 P15 P16 P17 P18
the molecule geometry in crystal form (Table 1). (Calculated) (Calculated) (Crystal)
Heat of Formation
−14.766 −25.638 −36.512 −47.387 −58.264 −69.141 −44.786 −37.285 -
[kcal/mol] Table 1. HOF, bond lengths and angles for P13–P18, BDDAC and BDDAB.
Distance [Å] Compound
N(2)... X− BDDAC BDDAB 3.535 BDDAB
1 2 3
3.137 3.137
P13 3.137
P14 3.137
P15 P16 3.137P17 3.137
P18 3.148 4.301
N(2)–C(1) 1.508 1.508 1.508 1.508 1.508 1.508 (Calculated)
1.508 (Calculated)
1.515 (Crystal) 1.517
Heat of Formation
N(2)–C(4) 1.502 1.502
−14.766 1.502
−25.638 1.502 −47.3871.502
−36.512 −58.264 1.502
−69.141 1.502
−44.786 1.510
−37.285 - 1.531
[kcal/mol]
N(2)–C(3) 1.536 1.536 1.536 1.536 1.536 1.536 1.537 1.539 1.546
Distance [Å]
N(2)–C(5) 1.529 1.529 1.529 1.529 1.529 1.529 1.528 1.534 1.510
N(2)...X− 3.137 3.137 3.137 3.137 3.137 3.137 3.148 3.535 4.301
C(5)–C(6) N(2)–C(1)
1.525 1.525
1.508
1.525
1.508
1.525 1.508 1.5251.508
1.508
1.525
1.508
1.525
1.508 1.515
1.526 1.517
1.538
C(3)–C(13) N(2)–C(4)1.494 1.494
1.502 1.494
1.502 1.494
1.502 1.502 1.4941.502 1.494
1.502 1.494
1.502 1.5101.492 1.531 1.487
C(13)–C(14) N(2)–C(3)1.397 1.397
1.536 1.397
1.536 1.397 1.536 1.3971.536
1.536 1.397
1.536 1.397
1.537 1.5391.397 1.546 1.402
C(14)–C(15) N(2)–C(5)1.394 1.394
1.529 1.394
1.529 1.394 1.529 1.3941.529
1.529 1.394
1.529 1.390
1.528 1.5341.389 1.510 1.393
C(15)–N(16) C(5)–C(6)1.353 1.353
1.525 1.353
1.525 1.353 1.525 1.3531.525
1.525 1.353
1.525 -
1.525 1.526 - 1.538 -
N(16)–C(17) C(3)–C(13)1.352 1.352
1.494 1.352
1.494 1.352 1.494 1.3521.494
1.494 1.352
1.494 -
1.494 1.492 - 1.487 -
C(17)–C(18) C(13)–C(14) 1.397 1.397
1.397 1.397
1.397 1.397
1.397 1.397 1.3971.397 1.397
1.397 1.397
1.391 1.3971.392 1.402 1.356
C(13)–C(18) C(14)–C(15) 1.396 1.394
1.396 1.394
1.396 1.394
1.396 1.394 1.3961.394 1.394
1.396 1.390
1.397 1.3891.398 1.393 1.363
C(15)–N(16) 1.353 1.353 1.353 1.353 1.353 1.353 - - -
Angle [◦ ] N(16)–C(17) 1.352 1.352 1.352 1.352 1.352 1.352 - - -
C(1)–N(2)–C(4)C(17)–C(18)108.556 1.397
108.558 1.397
108.558 1.397
108.560 1.397108.558
1.397 108.558
1.397 1.391
108.556 1.392
109.668 1.356 109.560
C(13)–C(18) 1.396 1.396 1.396 1.396 1.396 1.396 1.397 1.398 1.363
C(1)–N(2)–C(3) 107.159 107.159 107.157 107.158 107.158 107.159 107.223 108.079 108.440
Angle [°]
C(1)–N(2)–C(5) 110.034 110.035 110.034 110.034 110.035 110.035 109.989 108.868 110.100
C(1)–N(2)–C(4) 108.556 108.558 108.558 108.560 108.558 108.558 108.556 109.668 109.560
C(3)–N(2)–C(4) 110.028 110.024 110.024 110.024 110.024 110.023 109.982 112.142 108.160
C(1)–N(2)–C(3) 107.159 107.159 107.157 107.158 107.158 107.159 107.223 108.079 108.440
C(3)–N(2)–C(5) 110.391
C(1)–N(2)–C(5)
110.393
110.034
110.395
110.035
110.392110.034
110.034
110.393
110.035
110.393
110.035
110.446
109.989
109.140
108.868 110.100
112.530
C(4)–N(2)–C(5) 110.593
C(3)–N(2)–C(4) 110.593
110.028 110.594
110.024 110.593110.024
110.024 110.594
110.024 110.593
110.023 110.566
109.982 108.890
112.142 108.160 107.990
N(2)–C(3)–C(13) 114.561
C(3)–N(2)–C(5) 114.562
110.391 114.564
110.393 114.562110.392
110.395 114.562
110.393 114.561
110.393 114.413
110.446 113.849
109.140 112.530 116.070
N(2)–C(5)–C(6) 112.170
C(4)–N(2)–C(5) 112.171
110.593 112.171
110.593 112.171110.593
110.594 112.171
110.594 112.170
110.593 112.243
110.566 112.948
108.890 107.990 116.240
N(2)–C(3)–C(13) 114.561 114.562 114.564 114.562 114.562 114.561 114.413 113.849 116.070
Dihedral [◦ ]
N(2)–C(5)–C(6) 112.170 112.171 112.171 112.171 112.171 112.170 112.243 112.948 116.240
N(2)–C(3)–C(13)–C(14)
Dihedral 90.143
[°] 90.145 90.140 90.145 90.139 90.146 90.138 89.914 89.500
N(2)–C(3)–C(13)–C(18) −93.233
N(2)–C(3)–C(13)–C(14) −93.232
90.143 − 93.240
90.145 −93.23390.145
90.140 −93.241
90.139 −93.233
90.146 − 93.370
90.138 −93.929
89.914 89.500 −89.540
N(2)–C(3)–C(13)–C(18)
C(4)–N(2)–C(3)–C(13) 58.616 −93.233
58.621 −93.232
58.630 −93.240
58.620−93.23358.632
−93.241 58.626
−93.233 −93.370
60.176 −93.929
44.503 −89.540 60.950
C(4)–N(2)–C(3)–C(13)
C(5)–N(2)–C(3)–C(13) −63.720 58.616
−63.715 −58.621
63.707 58.630
−63.71558.620
−63.70458.632 −63.709
58.626 −60.176
62.133 44.503
−76.241 60.950 −58.260
C(5)–N(2)–C(3)–C(13)
C(1)–N(2)–C(3)–C(13) 176.460 −63.720
176.463 −63.715
176.471 −63.707
176.465−63.715 −63.704 176.469
176.474 −63.709 −62.133
178.030 −76.241
165.504 −58.260 179.690
C(1)–N(2)–C(3)–C(13) 176.460 176.463 176.471 176.465 176.474 176.469 178.030 165.504 179.690
1 benzyldimethyldodecylammonium chloride; 2 3
1 benzyldimethyldodecylammonium chloride; 2benzyldimethyldodecylammonium bromide;
benzyldimethyldodecylammonium bromide; 3 Ref. [35]. Ref. [35].

Figure 2. Representative molecular models of compounds P13, P15 and P17.

Figure 2. Representative molecular models of compounds P13, P15 and P17.
This allows one to extrapolate some conclusions from the calculated structure to the condensed
phase. The heat of formation (HOF) of the synthesized compounds decreases linearly with an increasing
This allows one to extrapolate some conclusions from the calculated structure to the condensed
number of methylene groups in the hydrocarbon chain (Figure 3a). The higher the number of methylene
phase. The heat
units, of formation
the lower (HOF) of
the heat of formation. theissynthesized
There compounds
also a good correlation betweendecreases linearly
the melting points of with an
increasingN,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium
number of methylene groups in the hydrocarbon chain
chlorides (Figure
and the number3a). The higher
of methylene the number
groups
in their alkyl chains (Figure 3a). The higher melting points reflect the better
of methylene units, the lower the heat of formation. There is also a good correlation between thepacking in the crystal
what is a result of the strong hydrophobic interactions between straight hydrocarbon chains. An
melting points of N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides and the number of
observed increasing stability of N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides
methylenewith
groups in their alkyl
the lengthening of thechains (Figure
alkyl chain is in a3a).
goodThe higherwith
accordance melting points reflect
the increasing melting the better
points of packing
in the crystal
thesewhat is a result
compounds, whichof the astrong
reflect sigmoidalhydrophobic interactions
type of correlation (Figure 3b).between straight hydrocarbon
chains. An observed increasing stability of N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium
chlorides with the lengthening of the alkyl chain is in a good accordance with the increasing melting
points of these compounds, which reflect a sigmoidal type of correlation (Figure 3b).
Molecules 2017, 22, 130 5 of 12
Molecules 2017, 22, 130 5 of 12

-10 HOF 105

-20 melting points 100

95

HOF (kcal/mol)
-30
90
-40

t (oC)
85
-50
80
-60
75
-70 70
-80 65
8 10 12 14 16 18
Alkyl chain length
(a) (b)
Figure 3. Plot (a) The relationships between calculated HOF and measured melting points vs. the length
Figure 3. Plot (a) The relationships between calculated HOF and measured melting points vs. the
of the hydrocarbon chain of compounds P13–P18; Plot (b) The correlation of melting points vs. HOF
length of
ofthe hydrocarbon chain of compounds P13–P18; Plot (b) The correlation of melting points vs.
P13–P18.
HOF of P13–P18.
2.3. Aggregation Behavior
2.3. Aggregation Behavior behavior of the synthesized surfactants was evaluated by a conductometric study
The aggregation
performed in aqueous solutions at 25 °C. Critical micellization concentrations (CMCs) for compounds
The aggregation behavior of the synthesized surfactants was evaluated by a conductometric study
P14–P18 were obtained from the plots of specific conductivity (κ) against the concentrations of the
performed in aqueous solutions ◦ C. Critical micellization concentrations (CMCs) for compounds
at 25 of
surfactants. A common method estimation of CMC values from conductivity vs. surfactant
were obtained
P14–P18concentration plotsfrom
is by the plotstheofintersection
finding specific conductivity
point of two (κ) leastagainst
squaresthe concentrations
method fitted lines of the
assigned
surfactants. A for pre- and method
common post-micellar regions. Unfortunately,
of estimation of CMC this method
values is not
from suitable for allvs.
conductivity typessurfactant
of
surfactants, as in some cases an inflection point is very difficult to find because of the
concentration plots is by finding the intersection point of two least squares method fitted lines assigned low curvature
of the plots. In such cases, the measurements are subject to large errors. In the case of the synthesized
for pre- and post-micellar regions. Unfortunately, this method is not suitable for all types of surfactants,
compounds, their CMCs were difficult to estimate by the conventional method. For that reason
as in some cases
critical an inflection
micellization point isforvery
concentrations difficult
compounds to find
P14–P18 because
were of by
obtained thethelow curvature
analysis of the of the
plots. In such
plots cases, theconductivity
of differential measurements (dκ/dc)are vs. subject
surfactanttoconcentration.
large errors.This Inisthe case
more of the
precise synthesized
approach
compounds, their
that has beenCMCs were difficult
successfully applied intodetermining
estimate by CMCthevalues
conventional
[36]. method. For that reason critical
micellization The plots of dκ/dc vs.
concentrations forconcentration
compounds possess reverse
P14–P18 weresigmoid shapesby
obtained andthe
thus can be fitted
analysis to aplots of
of the
Boltzmann-type sigmoid expressed by the following equation:
differential conductivity (dκ/dc) vs. surfactant concentration. This is more precise approach that has
been successfully applied in determining CMC −[36].
= values
+ (1)
1 +
The plots of dκ/dc vs. concentration possess reverse sigmoid shapes and thus can be fitted to a
Boltzmann-type sigmoid
Parameters expressed
A1 and by thethe
A2 represent following equation:
pre-micellar and post-micellar slopes, respectively. The
width of transition, dx, possesses a central point, x0, corresponding to the CMC value (Figure 4). CMC
values for compounds P14–P18 are given in Table 2.AP14–P17 − A2 display CMC values typical of cationic
y = A 2 + 1 x − x0 (1)
surfactants that decrease significantly with increasing alkyldx
chain length, because of the increase of the
1+e
hydrophobic character of the surfactants. In the case of P18 the increase of alkyl chain length causes a
less significant
Parameters change
A1 and A2inrepresent
CMC values
theprobably because
pre-micellar of coiling
and of the long
post-micellar alkyl chains
slopes, in water [37].
respectively. The width
of transition, dx, possesses a central point, x0 , corresponding to the CMC value (Figure 4). CMC values
Table 2. CMC values and Klevens equation parameters of P14–P18, benzalkonium chlorides (BAC)
for compounds P14–P18 are given
and trimethylammonium in (TMAC).
chlorides Table 2. P14–P17 display CMC values typical of cationic
surfactants that decrease significantly with increasing alkyl chain length, because of the increase
CMC [mM] Klevens Equation Parameters
of the hydrophobic character of the surfactants. In the case of P18 the increase of 2alkyl chain length
N1 10 12 14 16 18 A B R
causes a less significant
P14–P18 change
87.12 in CMC4.87
20.17 values1.14
probably
0.310 because
1.9978of coiling
0.3073 of the long alkyl chains in
0.9996
water [37]. TMAC 68.0 2 20.0 2 4.50 2 1.50 3 0.35 3 1.6927 0.2851 0.9983
BAC 39.0 2 8.8 2 2.00 2 0.49 4 0.093 5 1.8487 0.325 0.9994
Table 2. CMC values and
1 N = Klevens
alkyl chainequation parameters
length; 2 Ref. [37]; 3 Ref. of
[38]; 4 Ref. [39];benzalkonium
P14–P18, 5 Ref. [40]. chlorides (BAC)
and trimethylammonium chlorides (TMAC).

CMC [mM] Klevens Equation Parameters

N 1 10 12 14 16 18 A B R2
P14–P18 87.12 20.17 4.87 1.14 0.310 1.9978 0.3073 0.9996
TMAC 68.0 2 20.0 2 4.50 2 1.50 3 0.35 3 1.6927 0.2851 0.9983
BAC 39.0 2 8.8 2 2.00 2 0.49 4 0.093 5 1.8487 0.325 0.9994
1 N = alkyl chain length; 2 Ref. [37]; 3 Ref. [38]; 4 Ref. [39]; 5 Ref. [40].
Molecules 2017, 22, 130 6 of 12
Molecules 2017, 22, 130 6 of 12

CMC = x0 = 0.00114 mol/dm3

0.00150 1.0
Molecules 2017, 22, 130 6 of 12

0.00125 0.9
CMC = x0 = 0.00114 mol/dm3
0.00150 1.0
0.00100 0.8

/mol] [S*dm /mol]

0.00125 0.9

κ [S/dm]

2
0.00075 0.7
0.00100 x0 0.8

dκ/dc [S*dm 2dκ/dc

0.00050 0.6
κ [S/dm]

0.00075 0.7
0.00025 x0 0.5
0.00050 0.6
0.00000 0.4
0.00025 0.0004 0.0008 0.0012 0.0016 0.00200.5
C (mol/dm3)

Figure
Figure 4. Specific
4. Specific conductivity(•(•)
conductivity anddifferential
) and differential conductivity
0.00000
conductivityBoltzman-type fitting
Boltzman-type (―) (—)
fitting vs. concentration
vs. concentration
0.4

of P17 at
◦ 25 °C 0.0004 0.0008 0.0012 0.0016 0.0020
of P17 at 25 C. C (mol/dm )3

The
Figurenovel compounds
4. Specific possess
conductivity (•) andCMCs much
differential higher than
conductivity benzalkonium
Boltzman-type fitting chlorides bearing alkyl
(―) vs. concentration
The novel
chains ofof
P17the compounds
at 25 °C length. possess
same The dataCMCs obtained muchfromhigher
Figure 5thanand benzalkonium
Table 2 indicate chlorides bearing alkyl
that the compounds
presented
chains of the samein thislength.
paper exhibit
The datamicellization
obtainedbehavior more similar
from Figure 5 and to that 2ofindicate
Table trimethylalkylammonium
that the compounds
chlorides
presented The rather
novel
in this thanexhibit
to BACs.
compounds
paper The Klevens
possess CMCsbehavior
micellization muchequation
higher(2)than
more issimilar
often used
benzalkonium
to thatto ofcharacterize the relation
chlorides bearing alkyl
trimethylalkylammonium
chlorides rather than to BACs. The Klevens equation (2) is often used to characterize the the
between
chains ofthe
the CMC
same of a surfactant
length. The and
data its structure
obtained from [41].
FigureIt presents
5 and a
Table linear
2 relationship
indicate that thebetween
compoundsrelation
log CMC and alkyl
presented
between the CMC in this achain
ofpaper lengthmicellization
exhibit
surfactant of the surfactant:
and behavior[41].
its structure moreItsimilar
presents to that of trimethylalkylammonium
a linear relationship between the
chlorides rather than to BACs. The Klevens equation (2) is often used to characterize the relation
log CMC and alkyl chain length of the surfactant: logCMC = A − BN (2)
between the CMC of a surfactant and its structure [41]. It presents a linear relationship between the
where
log CMC A isanda constant that length
alkyl chain reflectsofthethefree energy change related to transfer of hydrophilic part of the
surfactant:
surfactant from the aqueous phase to the logCMC A − BN
micelle,=whereas parameter B determines the free energy (2)
logCMC = A − BN
change associated with the transfer of a methylene (−CH2−) unit of the hydrophobic chain from aqueous
(2)
where A is aAinto
solution
where constant
is a the thatthat
micelle.
constant reflects
is thethe
Nreflects thefree
number freeenergy
of carbon
energy change related
atomsrelated
change in alkyl toto transfer
chain
transfer of hydrophilic
of surfactant.
of hydrophilic part part
of theof the
surfactant from the aqueous phase to the micelle, whereas parameter
surfactant from the aqueous phase to the micelle, whereas parameter B determines the free energy B determines the free energy
change associated
change associated with 2.1the
with thetransfer
transfer ofofa methylene
a methylene (−CH(− 2−)CH
unit 2−of)the
unit of the hydrophobic
hydrophobic
BAC
chain from
chain from aqueous
aqueous solution
solution into the into the micelle.
micelle.
1.7 N is theN is
numberthe number
of carbon of carbon
atoms in atoms
alkyl in
chain alkyl
of chain
surfactant. of surfactant.
TMAC
1.3 P14-P18
2.1
BAC
0.9
1.7
Log CMC Log CMC

TMAC
0.5
1.3 P14-P18
0.1
0.9
-0.3
0.5
-0.7
0.1
-1.1
-0.3
8 10 12 14 16 18 20
-0.7 N
-1.1
Figure 5. Graphical presentation of the Klevens equation for P14–P18, TMAC and BAC (N = alkyl
8 10 12 14 16 18 20
chain length).
N

The value
Figure of B obtained
5. Graphical from linear
presentation fitting of theequation
Klevens equation (Table 2) allowed us(Nto=determine
Figure 5. Graphical presentation ofofthe
the Klevens
Klevens equationfor
forP14–P18,
P14–P18,TMAC
TMAC andand
BACBAC alkyl
(N = alkyl
the change
chain of the Gibbs free energy of methylene group transfer, ΔG(−CH2−), according to the equation:
length).
chain length).
ΔG( ) = −2.3RTB (3)
The value of B obtained from linear fitting of the Klevens equation (Table 2) allowed us to determine
The value of
the change ofBthe
obtained from
Gibbs free linear
energy offitting of the
methylene Klevens
group equation
transfer, (Table
ΔG(−CH2−) 2) allowed
, according to theusequation:
to determine
the change of the Gibbs free energy of methylene group transfer, ∆G(−CH2−) , according to the equation:
ΔG( ) = −2.3RTB (3)

∆G(−CH2−) = −2.3RTB (3)

Molecules 2017, 22, 130 7 of 12

where R is the universal gas constant and T is the absolute temperature. The Gibbs free energy
of methylene group transfer equals −1.71 kJ/mol, −1.63 kJ/mol and −1.85 kJ/mol for P14–P18,
trimethylalkylammonium chlorides and benzalkonium chlorides, respectively. The lower the Gibbs
free energy ∆G(−CH2−) the easier the process of micellization. Alkyl chain transfer from the aqueous
phase into the interior of the micelle reduces the energy of the solution, which promotes micellization.
However, within the micelles electrostatic repulsion exists between the positively charged molecules
of the surfactants, which hinders the micellization. Consequently, the CMC value depends on the
balance between the factors supporting and counteracting micellization. Therefore the hydrophilic
head structure it is an important factor determining the phenomenon of micellization. Benzalkonium
chlorides having within their hydrophilic part benzene rings lowering the polarity of the whole
molecule, possess much lower CMC values than trimethylalkylammonium chlorides. The newly
prepared salts have a pyridine ring that possesses a dipole moment and is incorporated into their
hydrophilic part. The presence of an electronegative nitrogen atom having a lone pair of electrons may
contribute to the additional repulsive interactions of the hydrophilic part of the surfactant, resulting in
increased CMC values as compared with the benzalkonium compounds.

2.4. Antimicrobial Properties

Minimal inhibitory concentrations (MIC) of N,N-dimethyl-N-(4-methylpyridyl)-N-alkyl-ammonium
chlorides have been determined against microscopic fungi, A. niger, C. albicans and P. chrysogenum as
well as bacteria, S. aureus, B. subtilis, E. coli and P. aeruginosa (Table 3). Since the octadecyl derivative
of the title compounds was not sufficiently soluble in water, therefore only the octyl, decyl, dodecyl,
tetradecyl and hexadecyl derivatives were tested.

Table 3. Antimicrobial properties of P13–P17.

MIC [mM]
Compound
A. niger C. albicans P. chrysogenum S. aureus B. subtilis E. coli P. aeruginosa
P13 - 12.497 - 6.2485 6.2485 6.2485 6.2485
P14 12.499 3.125 12.4999 1.5625 1.5625 1.5625 1.5625
P15 1.5625 0.7182 1.5625 0.1953 0.1953 0.3906 0.7812
P16 0.3906 0.1953 0.1953 0.0488 0.0977 0.1953 0.1953
P17 0.1953 0.0976 0.1953 0.0244 0.0244 0.0488 0.09764

The MIC values of N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides for fungi

range from 0.1 to 12 mM. The highest MIC values are obtained for the derivatives with the shortest
alkyl chains. As the length of the hydrocarbon chain increases the MIC in general decreases
(Figure 6). The lowest MIC values, i.e., the highest antifungal activity, are observed for the
hexadecyl derivatives. Aspergillus niger is usually more resistant to chemical disinfectants than other
microscopic fungi and is very difficult to eradicate from any medical or food industry environment.
The MIC values of N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides for all tested
fungi are similar, what means that decontamination of any area can be done using optimized
concentrations of N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides without the need
to use a high concentration of microbiocides to effectively remove of Aspergillus niger. MIC values
of pyridyl analogues of BAC for the bacteria S. aureus, B. subtilis, E. coli and P. aeruginosa are
lower than those for microscopic fungi and range from 6 to 0.02 mM (Table 3). Similarly to the
effects observed for microscopic fungi, the MIC values of alkyl derivatives for bacteria decrease
with the increasing length of the hydrocarbon chain (Figure 7). The antimicrobial activity of
alkylammonium salts is generally dependant on the length of hydrocarbon chain what is connected
with the potential to penetrate the cell wall. For benzalkonium chlorides and similar compounds
the lowest MIC values are observed for dodecyl and tetradecyl derivatives (Table 4). The shorter
hydrocarbon chain is not able to penetrate the cell wall; the longer substituent is too flexible that
is why it cannot also infiltrate the cell wall. The relationships between MIC and the number of
Molecules 2017, 22, 130 8 of 12

carbon atoms in the alkyl substituent are usually of parabolic type, with minima around C12-C14.
For N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides the lowest MIC values are
Molecules 2017, 22, 130 8 of 12
observed for
Molecules C16,
2017, i.e., at a slightly higher chain length than for benzalkonium chlorides.8 ofBecause
22, 130 12
of thevalues
low solubility
are observed of for
longer
C16, alkyl
i.e., atderivatives we were
a slightly higher chain unable to check
length than their MIC and
for benzalkonium compare it
chlorides.
withvalues
BACs.are
Because of observed
Inthe
general for C16,ofi.e.,
benzalkonium
low solubility at aalkyl
longer slightly higher
chlorides show
derivatives chain length
weslightly
were than
to for
lower
unable MICbenzalkonium
check values
their MICin chlorides.
comparison
and compare to
Because
it with of the In
BACs. low solubility
general of longer alkyl
benzalkonium derivatives
chlorides show we were lower
slightly unableMIC
to check their
values in MIC and compare
comparison to the
the pyridyl analogues, however for Pseudomonas aeruginosa the C16 pyridyl analogue showed better
itpyridyl
with BACs. In general
analogues, benzalkonium
however chlorides show slightly lower MIC values in comparison to the
antimicrobial activity (Table 4). for Pseudomonas aeruginosa the C16 pyridyl analogue showed better
pyridyl analogues,
antimicrobial however
activity for Pseudomonas aeruginosa the C16 pyridyl analogue showed better
(Table 4).
antimicrobial activity (Table 4).
Table 4. Literature values of MIC of benzalkonium chlorides [42].
Table 4. Literature values of MIC of benzalkonium chlorides [42].
Table 4. Literature values of MIC of benzalkonium chlorides [42].
MICMIC
[mM][mM]
N1 N1 MIC [mM]
N 1 P.P.aeruginosa
aeruginosa A. A. niger
niger C. albicans
C. albicans
8 P. aeruginosa A. niger
5 5 C. albicans
8 55 2.5 2.5
10 8
10 5
1.25
1.25 5
0.625
0.625 2.5
0.312 0.312
12 10
12 1.25
0.312
0.312 0.625
0.0781
0.0781 0.312
0.039 0.039
14 12 0.0781
0.312 0.0195
0.0781 0.039 0.0097
14 0.0781 0.0195 0.0097
16 14 0.156
0.0781 0.156
0.0195 0.0097 0.0195
16 0.156 0.156
1 N = alkyl chain length.
0.0195
16 0.156 0.156
1 N = alkyl chain length.
0.0195
1 N = alkyl chain length.
13
13 12.4970 12.4999 1.5625
12 1.5 A. niger
12.4970 12.4999 1.5625
12
11 1.5 A.
C. niger
albicans
[mM]

11 C. albicans
[mM]

10 1 P. chrysogenum
MIC

109 1 P. chrysogenum
MIC

98 0.7182
[mM]

0.5 0.7182
87
[mM]

0.5 0.3906
76
MIC

0.3906
0.1953 0.1953
65 0.0976
MIC

0 0.1953 0.1953
0.0976
54 0 12 14 16
43 3.1250 12 14 16
32 3.1250
21
10
0 8 10 12 14 16
8 10 N
12 14 16
N
Figure
Figure 6. 6. Antifungalactivity
Antifungal activity of
of P13–P17
P13–P17(N
(N= =
alkyl chain
alkyl length).
chain length).
Figure 6. Antifungal activity of P13–P17 (N = alkyl chain length).
0.8 0.7812
6.2485 S. aureus
0.8
0.7 0.7812
6 S.
B.aureus
subtilis
6.2485
6 0.7
0.6 B.
E. subtilis
coli
0.6
[mM]

5 0.5 E.
P. coli
aeruginosa
[mM]

5 0.5
0.4 P. aeruginosa
0.3906
MIC

4 0.4
0.3 0.3906
MIC
[mM]

4 0.3
0.2 0.1953
0.1953
[mM]

0.2 0.1953 0.1953

0.0977 0.0976
3 0.1
MIC

0.10 0.0488
0.0977 0.0488
0.0976
3
MIC

0.0244
0.0488
0.0488
2 0 12 14 16 0.0244
2 1.5625 12 14 16

1 1.5625
1
0
0 8 10 12 14 16
8 10 N
12 14 16
N
Figure 7. Antibacterial activity of P13–P17 (N = alkyl chain length).
Figure
Figure 7. 7. Antibacterialactivity
Antibacterial activity of
ofP13–P17
P13–P17(N(N
= alkyl chain
= alkyl length).
chain length).
Molecules 2017, 22, 130 9 of 12

3. Materials and Methods

3.1. General Information

The NMR spectra were measured in CDCl3 as a solvent with a 300 MHz Mercury NMR
spectrometer (Varian, Oxford, UK). Infrared spectra of P13, P15, P16 and P18 were recorded as
KBr pellets using a IFS 66 FT-IR spectrometer (Bruker, Karlsruhe, Germany) at 295 K. Infrared
spectra of P14 and P17 were recorded on a ATR 4700 type FT-IR spectrometer (Jasco, Easton, MD,
USA). The Electron Spray Ionization (ESI) mass spectra were recorded in methanol on a ZQ mass
spectrometer (Waters/Micromass, Manchester, UK) equipped with a syringe pump (Harvard Apparatus,
St. Laurent, QC, Canada). N,N-dimethylamines were purchased from Aldrich (Poznań, Poland) except for
N,N-dimethyloctadecylamine that was purchased from TCI (Łódź, Poland). 4-Chloromethylpyridine (6)
was obtained from 4-chloromethylpyridine hydrochloride (purchased from Alfa Aesar, Karlsruhe,
Germany) according to a literature procedure [43] and used further without purification. Organic
solvents were purchased from POCH (Gliwice, Poland) except for acetonitrile that was purchased from
VWR (Gdańsk, Poland).

3.2. Typical Procedure of Synthesis of N,N-Dimethyl-N-(4-methylpyridyl)-N-alkylammonium

Chlorides P13–P18
A mixture of 4-chloromethylpyridine (6, 1 g, 7.87 mmol) and an N,N-dimethylalkylamine
(7.87 mmol) in CH3 CN (10 mL) was stirred at room temperature for 24 h. Completion of reaction was
controlled by TLC (CHCl3 :MeOH 5:1). Then the solvent was evaporated under reduced pressure and
the residue was washed twice with diethyl ether. The crude dark brown product was purified by
recrystallization from acetone/acetonitrile (1:1).
N,N-Dimethyl-N-(4-methylpyridyl)-N-octylammonium chloride (P13). Light brown solid, yield: 79%;
m.p. 67–70 ◦ C; IR (KBr) νmax 3057, 2929, 2850, 1596, 1473 cm−1 ; 1 H-NMR (CDCl3 ) δ 8.71 (2 H, d,
J = 5.9 Hz, H-a), 7.73 (2 H, d, J = 6.0 Hz, H-b), 5.24 (2 H, s, H-d), 3.53 (2 H, m, H-f), 3.34 (6 H, s, H-e),
1.80 (2 H, m, H-g), 1.15–1.39 (10 H, m, H-h), 0.87 (3 H, t, J = 6.6, 6.9 Hz, H-i); 13 C-NMR (CDCl3 ) δ 150.65
(CH, C-a), 136.17 (C, C-c), 127.59 (CH, C-b), 65.58 (CH2 , C-d), 64.06 (CH2 , C-f), 49.86 (CH3 , C-e), 31.47
(CH2 , C-g), 29.05–22.43 (CH2 , C-h), 13.92 (CH, C-d); ESI-MS m/z: 249 [M]+ .
N,N-Dimethyl-N-(4-methylpyridyl)-N-decylammonium chloride (P14). Light orange solid, yield: 76%;
m.p. 72–75 ◦ C; IR (KBr) νmax 3023, 2921, 2850, 1601, 1470 cm−1 ; 1 H-NMR (CDCl3 ) δ 8.71 (2 H, d,
emphJ = 5.9 Hz, H-a), 7.72 (2 H, d, J = 6.0 Hz, H-b), 5.26 (2 H, s, H-d), 3.53 (2 H, m, H-f), 3.34 (6 H, s,
H-e), 1.80 (2 H, m, H-g), 1.15–1.39 (10 H, m, H-h), 0.88 (3 H, t, J = 6.6, 6.9 Hz, H-i); 13 C-NMR (CDCl3 ) δ
150.67 (CH, C-a), 136.14 (C, C-c), 127.59 (CH, C-b), 65.57 (CH2 , C-d), 64.04 (CH2 , C-f), 49.86 (CH3 , C-e),
31.71 (CH2 , C-g), 29.27–22.53 (CH2 , C-h), 14.00 (CH3 , C-i); ESI-MS m/z: 277 [M]+ .
N,N-Dimethyl-N-(4-methylpyridyl)-N-dodecylammonium chloride (P15). Light brown solid, yield: 81%;
m.p. 83–86 ◦ C; IR (KBr) νmax 3062, 2920, 2850, 1602, 1470 cm-1 ; 1 H-NMR (CDCl3 ) δ 8.70 (2 H, d,
J = 5.9 Hz, H-a), 7.72 (2 H, d, J = 6.0 Hz, H-b), 5.25 (2 H, s, H-d), 3.52 (2 H, m, H-f), 3.34 (6 H, s, H-e),
1.80 (2 H, m, H-g), 1.15–1.39 (10 H, m, H-h), 0.88 (3 H, t, J = 6.6, 6.9 Hz, H-i); 13 C-NMR (CDCl3 ) δ 150.66
(CH, C-a), 136.14 (C, C-c), 127.58 (CH, C-b), 65.57 (CH2, C-d), 64.02 (CH2, C-f), 49.86 (CH3, C-e), 31.77
(CH2, C-g), 29.46–22.56 (CH2 , C-h), 14.00 (CH3, C-i); ESI-MS m/z: 305 [M]+ .
N,N-Dimethyl-N-(4-methylpyridyl)-N-tetradecylammonium chloride (P16). Light brown solid, yield: 83%;
m.p. 91–93 ◦ C; IR (KBr) νmax 3062, 2915, 2850, 1602, 1470 cm-1 ; 1 H-NMR (CDCl3 ) δ 8.71 (2 H, d,
J = 5.9 Hz, H-a), 7.73 (2 H, d, J = 6.0 Hz, H-b), 5.30 (2 H, s, H-d), 3.54 (2 H, m, H-f), 3.35 (6 H, s, H-e),
1.80 (2 H, m, H-g), 1.20–1.39 (10 H, m, H-h), 0.88 (3 H, t, J = 6.8, 7.3 Hz, H-i); 13 C-NMR (CDCl3 ) δ 150.68
(CH, C-a), 136.12 (C, C-c), 127.58 (CH, C-b), 65.49 (CH2, C-d), 64.02 (CH2, C-f), 49.79 (CH3, C-e), 31.80
(CH2, C-g), 29.53–22.58 (CH2, C-h), 14.03 (CH3, C-i); ESI-MS m/z: 333 [M]+ .
Molecules 2017, 22, 130 10 of 12

N,N-Dimethyl-N-(4-methylpyridyl)-N-hexadecylammonium chloride (P17). Light brown solid, yield: 85%;

m.p. 96–98 ◦ C; IR (KBr) νmax 3053, 2921, 2850, 1601, 1470 cm-1 ; 1 H-NMR (CDCl3 ) δ 8.71 (2 H, d,
J = 5.9 Hz, H-a), 7.73 (2 H, d, J = 6.0 Hz, H-b), 5.30 (2 H, s, H-d), 3.54 (2 H, m, H-f), 3.35 (6 H, s, H-e),
1.80 (2 H, m, H-g), 1.19–1.37 (10 H, m, H-h), 0.88 (3 H, t, J = 6.6, 6.9 Hz, H-i); 13 C-NMR (CDCl3 ) δ 150.70
(CH, C-a), 136.11 (C, C-c), 127.58 (CH, C-b), 65.51 (CH2, C-d), 64.04 (CH2, C-f), 49.80 (CH3, C-e), 31.83
(CH2, C-g), 29.61–22.59 (CH2, C-h), 14.04 (CH3, C-i); ESI-MS m/z: 361 [M]+.
N,N-Dimethyl-N-(4-methylpyridyl)-N-octadecylammonium chloride (P18). Light brown solid, 88%; m.p.
100–102 ◦ C; IR (KBr) νmax 3054, 2915, 2851, 1600, 1472 cm-1 ; 1 H-NMR (CDCl3 ) δ 8.71 (2 H, d, J = 5.9 Hz,
H-a), 7.73 (2 H, d, J = 6.0 Hz, H-b), 5.30 (2 H, s, H-d), 3.54 (2 H, m, H-f), 3.35 (6 H, s, H-e), 1.80 (2 H, m,
H-g), 1.17–1.35 (30 H, m, H-h), 0.87 (3 H, t, J = 6.4, 7.3 Hz, H-i); 13 C-NMR (CDCl3 ) δ 150.68 (CH, C-a),
136.12 (C, C-c), 127.58 (CH, C-b), 65.49 (CH2, C-d), 64.02 (CH2, C-f), 49.79 (CH3, C-e), 31.80 (CH2, C-g),
29.56–22.58 (CH2, C-h), 14.03 (CH3, C-i); ESI-MS m/z: 389 [M]+.

3.3. Antimicrobial Properties Evaluation

The MIC values of P13–P17 against bacteria and microscopic fungi (yeast and moulds) were
measured at the Institute of Fermentation Technology and Microbiology, Technical University of
Lodz in Poland. The MICs are defined as the lowest concentration of the compounds at which there
was no visible growth of microorganisms. MIC values were determined by a standard tube 2-fold
dilution method [44,45]. The experiments were carried out on the bacteria Escherichia coli ATCC 10536,
Pseudomonas aeruginosa ATCC 85327, Staphylococcus aureus ATCC 6538, Bacillus subtilis NCAM 01644 and
microscopic fungi Candida albicans ATCC 10231, Aspergillus niger ATCC 16404, Penicillium chrysogenum
ATCC 60739. Antifungal properties were checked on a Malt Extract Broth medium (Merck, Darmstadt,
Germany) at a density of inoculum of 1–2 × 106 cfu/mL; antibacterial activity—on Trypticase Soy
Broth (Merck), density of inoculum 1–2 × 107 cfu/mL. All tests were repeated three times.

3.4. Conductometric Study

Measurements of conductivity were obtaining using a CO300 conductivity meter (VWR, Gdańsk,
Poland). CMC values of compounds P14–P18 were obtained from conductometric titrations conducted
in double distilled water in 25 ◦ C. For P14–P16, to an initial volume of surfactant solution water was
added gradually, and after every addition the conductivity of the solution was measured. For P17 and
P18 to an initial volume of water surfactant solution was added gradually, and after every addition
conductivity of the solution was measured. For each compound, analysis was conducted twice and
CMC values reported in this work are the average of the two results.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/22/

1/130/s1.
Acknowledgments: This work has been supported by the National Centre for Research and Development (Poland;
TANGO1/266340/NCBR/2015).
Author Contributions: Izabela Małecka and Bogumił Brycki conceived and designed the experiments;
Izabela Małecka performed the chemical experiments; Anna Koziróg and Anna Otlewska performed the
microbiological experiments; Izabela Małecka and Bogumił Brycki analyzed the data and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds P13–P17 are available from the authors.

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