Synthesis and disinfection effect of the pyridine-4-aldoxime based salts

Article abstract of DOI:10.3390/molecules20033681

A set of new quaternary ammonium compounds based on pyridine-4-aldoxime was synthesized, characterized with analytical data (NMR, EA, HPLC, MS) and tested for in vitro antimicrobial activity (antibacterial, antifungal) and cytotoxicity. Quaternary pyridinium-4-aldoxime salts with length of alkyl side chain from C8 to C20 and belonging to the group of cationic surfactants were investigated in this work. An HPLC experimental protocol for characterization of mixtures of all homologues has been found. Antimicrobial evaluation found that yeast-type fungi were most sensitive towards C₁₄ and C₁₆ analogues, whereas the C₁₆ analogue was completely ineffective against filamentous fungi. Antibacterial assessment showed versatility of C₁₄ and relatively high efficacy of C₁₆ against G+ strains and C₁₄ against G- strains. Notably, none of the studied compounds exceeded the efficacy and versatility of the benzalkonium C₁₂ analogue, and benzalkonium analogues also exhibited lower cytotoxicity in the cell viability assay.

Full text of DOI:10.3390/molecules20033681

Molecules 2015, 20, 3681-3696; doi:10.3390/molecules20033681
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Disinfection Effect of the Pyridine-4-aldoxime
Based Salts
Jan Marek 1,2, David Malinak 3,4, Rafael Dolezal 3, Ondrej Soukup 3,5, Marketa Pasdiorova 2,3
,
Martin Dolezal 1 and Kamil Kuca 3,5,
*
1
Department of Medicinal Chemistry and Drug Analysis, Faculty of Pharmacy, Charles University in
Prague, Heyrovskeho 1203, Hradec Kralove 500 05, Czech Republic;
E-Mails: marekjanmgr@seznam.cz (J.M.); martin.dolezal@faf.cuni.cz (M.D.)
Department of Epidemiology, Faculty of Military Health Sciences, University of Defence,
Trebesska 1575, Hradec Kralove 500 01, Czech Republic; E-Mail: MarketaPasdiorova@seznam.cz
Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581,
Hradec Kralove 500 05, Czech Republic; E-Mails: david.malinak@gmail.com (D.M.);
rafael.dolezal@fnhk.cz (R.D.); soukup.ondrej07@gmail.com (O.S.)
2
3
4
5
Department of Cybernetics and Biomedical Engineering, Faculty of Electrical Engineering and
Computer Science, VSB-Technical University of Ostrava, 17. Listopadu 15, Ostrava-Poruba 708 33,
Czech Republic
Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University
of Defence, Trebesska 1575, Hradec Kralove 500 01, Czech Republic
* Author to whom correspondence should be addressed; E-Mail: kamil.kuca@fnhk.cz;
Tel.: +420-495-832-923.
Academic Editor: Peter J. Rutledge
Received: 4 January 2015 / Accepted: 12 February 2015 / Published: 24 February 2015
Abstract: A set of new quaternary ammonium compounds based on pyridine-4-aldoxime
was synthesized, characterized with analytical data (NMR, EA, HPLC, MS) and tested for
in vitro antimicrobial activity (antibacterial, antifungal) and cytotoxicity. Quaternary
pyridinium-4-aldoxime salts with length of alkyl side chain from C8 to C20 and belonging
to the group of cationic surfactants were investigated in this work. An HPLC experimental
protocol for characterization of mixtures of all homologues has been found. Antimicrobial
evaluation found that yeast-type fungi were most sensitive towards C14 and C16 analogues,
whereas the C16 analogue was completely ineffective against filamentous fungi.
Antibacterial assessment showed versatility of C14 and relatively high efficacy of C16

Molecules 2015, 20
against G+ strains and C14 against G− strains. Notably, none of the studied compounds
3682
exceeded the efficacy and versatility of the benzalkonium C12 analogue, and benzalkonium
analogues also exhibited lower cytotoxicity in the cell viability assay.
Keywords: pyridinium-4-aldoxime salts; synthesis; analysis; antimicrobial activity;
surfactants; cytotoxicity
1. Introduction
The 20th century can appropriately be called the age of organic chemistry. It brought many new
organic structures, both natural and synthetic. Here the discussed surfactants are undoubtedly a huge
group of organic compounds for which a technical use has been found. These compounds have
certainly undergone considerable development since their discovery in 1930, and countless
applications have been reported for these important molecules (e.g., detergents, disinfectants,
decontaminants, etc.) [1–3].
Cationic surfactants consist of a hydrophilic part, such as a quaternary nitrogen moiety, able to
interact with polar chemical milieu, and a hydrophobic part (e.g., a long alkyl chain), which can, on the
other hand, penetrate into non-polar molecular agglomerates.
Many types of cationic surfactant based on quaternary nitrogen have been described [4–6]. One
important property described for quaternary ammonium salts is inhibition of the growth of bacteria and
fungi. The most commonly used are derivatives of pyridinium, cetrimonium, benzalkonium and
benzoxonium salts [7–10]. Some pyridinium salts are already in use as disinfectants in many
preparations (as a component of eye drops, solutions, disinfection foams, etc.). Currently, there have
been many articles describing cetylpyridinium salt as a protective agent against food-borne disease [6].
Recently, polymeric quaternary ammonium salts have been used in the development of bactericidal
surfaces [11,12]. Another antimicrobial application of cetylpyridinium is its addition to chewing gums
or dental formulations as an antiplaque agent [13,14]. In order to intensify the development of
cetylpyridinium analogues, the 2D and 3D QSAR method was established [6]. It was found that
numerous properties such as substituents on the pyridine moiety, alkyl side-chain length, hydrophobicity,
pKa and absorbability on to the cell are critically important for antimicrobial activity [15,16].
Quaternary ammonium salts (QAS) are also frequently used in prevention of nosocomial infections
that are predominantly acquired within healthcare facilities, and conditioned mainly by the impaired
immune status of patients, absence of a strict glove-change regime, and the use of improperly or
inadequately sterilized medical instruments [17]. An important property of such compounds is their
ability to form micelles (Figure 1). These formations are created mainly in aqueous solution, when the
critical micelle concentration is exceeded. Many cationic surfactants can also be used as micellar
catalysts [18], able to accelerate chemical decomposition [19,20], and such compounds can find use as
decontaminants for military purposes. Thus these properties are important in decontamination
procedures for measuring the kinetics of cleavage of some model compounds such as
organophosphates and others [21–26]. Many other properties of cationic surfactants were discovered.
Enthalpies of dilution, density effects of counterions in micellar solution and osmotic coefficients have

Molecules 2015, 20
3683
been measured as a function of concentration, and the results expressed in terms of partial molar
quantities [27–29].
air
monolayer
soluble monomer
micelle
water
Figure 1. Micelles.
Our group has been devoted to the synthesis and evaluation of derivatives of heterocyclic nitrogen
compounds [30–33] and tensides [34–36]. Preparation of the various pyridinium-4-aldoxime salts has
already been described before by our group, where the compounds were evaluated as micellar
catalysts [24,25]. However, there has been no description of the synthesis of the whole series of such
salts differing in the alkyl chain (C8 to C20), and their antimicrobial properties have not been measured.
The primary aim of this work was to investigate this group of QAS as potential affordable
disinfectants. For this reason a universal method was developed for preparation of monoquaternary
pyridinium-4-aldoxime salts with alkyl-chain substituents, and the prepared compounds were characterized
using analytical data (NMR, EA, HPLC, MS) and tested for in vitro antimicrobial activity and cytotoxicity.
2. Results and Discussion
2.1. Synthesis and Analysis
The results achieved within our study are shown in Table 1. This includes yields, melting points,
retention times obtained on HPLC and calculated logP (ClogP). It is evident that the preparation of
pyridinium-4-aldoxime salts with side chain C8–C20 is a quite elementary one step reaction. On the
other hand, repeated crystallization was necessary in order to achieve the required purity. Two
different conditions for synthesis were used. The reactions were realized in two different solutions,
ethanol (EtOH) or acetonitrile (CH3CN) (Scheme 1), and the prepared compounds 9–15 were

Molecules 2015, 20
3684
recrystallized from ethyl acetate or acetone. There was no significant variation in the yields from both
solvents. Yields were highest for compounds 11–13. A comparison can be made of the influence of the
non-polar part of molecule on the yields during the quaternization of N,N-dimethyl-N-benzylamine,
pyridine or derivatives of quinoline [31,33,37,38]. As expected, the value of ClogP increases with the
length of the non-polar chain.
Table 1. Yields, melting points and retention times of prepared pyridinium-4-aldoxime salts.
Comp.
9
R
Yields (%) EtOH Yields (%) CH3CN
m.p. (°C)
92–93
HPLC Rt (min)
ClogP
0.04
1.10
2.15
3.21
4.27
5.33
6.39
C8
15
27
70
72
75
47
45
X
41
55
75
87
76
X
4.15
4.67
5.22
5.82
6.49
7.23
8.06
10
C10
C12
C14
C16
C18
C20
130–132
136–137
144–146
144–145
128–130
126–128
11
12
13
14
15
Note: X—Not prepared in CH3CN.
NOH
NOH
CH3CN or EtOH
Br
Br
+
n
N
reflux
48 hours
N
1
2-8
n = 6, 8, 10, 12, 14, 16, 18
9-15
Scheme 1. Preparation of pyridinium-4-aldoxime salts.
RT: 0.00 - 9.98
NL:
4.67
4.40E5
Channel B
UV
smes1_100
_01
400000
350000
300000
250000
200000
150000
100000
50000
5.82
4.15
5.22
6.49
7.23
8.06
1.78
8.99
9
2.47
1.40
9.68
0.63 1.07
1
2.89
3
3.88
0
0
2
4
5
6
7
8
Time (min)
Figure 2. HPLC chromatogram of pyridinium-4-aldoxime salts mixture.
Additionally, HPLC analysis was carried out for individual compounds. A newly developed HPLC
method was able to distinguish all prepared quaternary pyridinium-4-aldoxime salts (Figure 2). The

Molecules 2015, 20
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shortest retention time was found for the C8 pyridinium-4-aldoxime salt. This novel HPLC method
could be easily used for characterization of mixtures of all homologues.
2.2. Antimicrobial Activity
A set of eight fungus strains was used to evaluate antifungal activity. The minimum inhibitory
concentrations (MICs) and minimum fungicidal (MFCs) concentrations for four yeasts and four
filamentous fungi are summarized in Table 2. The whole set of pyridinium-4-aldoxime salts was tested
for biological activity. The results for compounds 9–15 as well as for the reference benzalkonium B12,
B14 and B16 compounds are shown in Table 2.
A broad spectrum of activity is evident for compounds 11–14. The CK strain is sensitive for the
whole set of pyridinium-4-aldoxime salts except for compound 15 (C20 alkyl chain). The yeast-type
strains (CA, CT, CK, CG) are most sensitive to compounds 12–14. The effectiveness is comparable
with the benzalkonium salts. However, there is no clear correlation with the alkyl chain length.
The disruptive effect of QAS on the microorganism is probably based on the adsorption of this
amphiphile molecule on the outer cellular membrane. The positively charged “heads” of the cationic
molecules interact with the negatively charged cell membrane, disrupting it thanks to electrostatic and
hydrophobic interactions. These interactions effectively out-compete the divalent cations, which
normally stabilize surface structures by linking adjacent negatively-charged components [39]. Once
close contact is accomplished by the hydrophilic region, the hydrophobic region proceeds to penetrate
the hydrophobic bilayer to cause cell leakage and lysis [40]. This cascade leads to the release of K+ and
cytoplasmic components, and finally to death of the cell. The antibacterial activity thus depends on the
hydrophilic-hydrophobic balance of the cationic surfactants. Increase in the alkyl chain length (from
C12 to C16) increases the hydrophobic character, which may be too high to facilitate transport through
the bacterial cell membrane [41].
The filamentous fungi strains have significant sensitivity only to compounds 12 and 13 (alkyl length
C14 and C16). The values of MICs and MFCs are higher compared to benzalkonium reference salts. The
other compounds have no or only minor efficacy against filamentous fungi.
The in vitro antibacterial activity of compounds 9–15 was also assayed for the eight strains of
bacteria. Gram positive (G+) and gram-negative (G−) bacteria groups are represented to cover the entire
spectrum. Listed in Table 3 are the obtained minimum inhibitory concentrations (MICs) after 24 h and
48 h of incubation, and the minimum bactericidal concentrations (MBCs) after 48 h of incubation.
Again, commercially used benzalkonium salts with alkyl side-chain length C12, C14 and C16 are
included for comparison of antibacterial activity (B12, B14, B16).
In general, the relationship of structure and antibacterial activity shows the most efficient
compounds to be 11–13 (alkyl chain C12, C14, C16), highlighting the versatility of 12 and the high efficacy
of 13 against G+ strains. The other compounds with a shorter or longer lipophilic chain did not show any
distinctive effect. Only the relatively high potency of 11, 14 and 15 against SE can be noted. It was found
that the Gram-positive bacteria (first four strains) are most sensitive to compounds 12 and 13 (alkyl chain
C14 and C16). In comparison with the benzalkonium salts, the effectiveness of compound 13 seems to
be better (MBC) against SA. The sensitivity of the other strains of G+ bacteria is comparable or lower.

Molecules 2015, 20
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Table 2. Minimum inhibitory/fungicidal concentrations of the prepared compounds (9–15) and selected derivatives of N-benzalkonium
(B12–B16). The in vitro activities were determined on a panel of eight fungal strains.
MIC (μmol/L); 24 h/48 h Incubation
MFC (μmol/L); 48 h Incubation
Microorganisms
a
a
a
9
10
125/250
250
11
12
13
3.9/3.9
3.9
14
15
B12
B14
B16
Candida albicans ATCC
>500/>500
>500
15.62/15.62 1.95/1.95
7.81/7.81 >500/>500 0.49/0.49 7.81/7.81 3.91/7.81
15.62 >500 0.98 125 7.81
7.81/7.81 >500/>500 0.49/0.49 3.91/3.91 3.91/7.81
44859 (CA)
15.62
1.95
3.9/3.9
15.62
125/125
250
31.25/31.25 7.81/7.81
3.9/3.9
3.9
Candida tropicalis 156 (CT)
Candida krusei E28 (CK)
Candida glabrata 20/I (CG)
125
3.9/3.9
15.62
31.25
0.98/1.95
3.9
15.62
3.9/3.9
15.62
3.9/3.9
7.81
>500
>500/>500 0.49/0.49 3.91/3.91 1.95/1.95
>500 0.49 125 1.95
>500/>500 0.49/0.49 7.81/7.81 1.95/3.91
>500 0.49 125 62.5
0.98
125
7.81
31.25/62.5
125
1.95/1.95
15.62
3.9/ 3.9
3.9
125/125
250
31.25/31.25
62.5
3.9/3.9
3.9
1.95/3.9
31.25
3.9/3.9
3.9
Trichosporon asahii
1188 (TA)
>500 >500 500/>500
>500 >500
>500/>500 >500/>500
>500 >500
>500/>500 >500/>500
>500 >500
>500>500 >500/>500
>500 >500
125/125
500
15.62/15.62 7.81/7.81
31.25 7.81
62.5/62.5 15.62/15.62 >500/>500 >500/>500 0.98/3.91 7.81/15.62 7.81/7.81
250 62.5 >500 >500 3.91 125 62.5
62.5/62.5 15.62/31.25 >500/>500 >500/>500 7.81/7.81 31.25/31.25 7.81/7.81
500 62.5 >500 >500 7.81 125 62.5
15.62/62.5 15.62/15.62 >500/>500 >500/>500 0.98/0.98 15.62/15.62 7.81/7.81
125 62.5 >500 >500 1.95 15.62 62.5
7.81/7.81 >500/>500 0.49/1.95 31.25/31.25 7.81/7.81
7.81 >500 1.95 125 62.5
Aspergillus fumigatus
231 (AF)
125/250
>500
Absidia corymbifera
272 (AC)
500/500
500
Trichophyton
62.5/62.5
125
mentagrophytes 445 (TM)
a
Notes:
B12, B14, B16 mean N-benzyl-N,N-dimethyl-N-dodecylammonium bromide, N-benzyl-N,N-dimethyl-N-tetradecylammonium bromide, N-benzyl-N,
N-dimethyl-N-hexadecylammonium bromide, respectively. The preparation [33] and antimicrobial efficacy of B12–16 has been published elsewhere. The antifungal
activities against Trichophyton mentagrophytes 445 were determined after 72 h and 120 h of incubation.

Molecules 2015, 20
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Table 3. Minimum inhibitory/bactericidal concentrations of the prepared compounds (9–15) and selected derivatives of N-benzalkonium
(B12–B16). The in vitro activities were determined on a panel of eight bacterial strains.
MIC (μmol/L); 24 h/48 h Incubation
MBC (μmol/L); 48 h Incubation
Microorganisms
a
a
a
9
10
11
12
13
14
15
15.62/15.62
15.62
B12
B14
B16
Staphylococcus aureus
CCM 451608 (SA)
Staphylococcus aureus H
599608 (MRSA)
125/ 125
125
15.62/15.62 15.62/15.62
1.95/7.81
7.81
0.98/0.98
0.98
7.81/7.81
7.81
0.49/1.95
1.95
0.98/0.98
3.91
0.98/0.98
3.91
15.62
31.25/31.25
31.25
15.62
7.81/15.62
15.62
3.9/15.62
15.62
0.49/3.9
7.81
0.98/7.81
7.81
15.62/125
125
62.5/>250
>250
0.49/0.49
0.98
1.95/1.95
3.91
1.95/1.95
3.91
Staphylococcus epidermidis
H 696608 (SE)
62.5/125
250
15.62/15.62
15.62
0.98/1.95
1.95
0.98/0.98
0.98
0.98/0.98
0.98
1.95/1.95
1.95
1.95/1.95
1.95
0.49/0.49
0.49
0.98/0.98
0.98
0.49/0.49
3.91
Enterococcus sp.
500/>500
>500
62.5/125
500
31.25/31.25
62.5
0.98/7.81
7.81
7.81/7.81
7.81
3.9/15.62
15.62
62.5/62.5
62.5
0.49/0.98
0.98
1.95/1.95
7.81
1.95/1.95
3.91
J 1436508 (ES)
Escherichia coli
>500/>500
>500
500/500
500
62.5/62.5
62.5
15.62/15.62 15.62/15.62
15.62 15.62
15.62/15.62 15.62/15.62
15.62 15.62
15.62/15.62 31.25/31.25
>500/>500
>500
>250/>250
>250
0.49/1.95
1.95
7.81/7.81
7.81
7.81/7.81
7.81
CCM 4517 (EC)
Klebsiella pneumoniae D
1175008 (KP)
>500/>500
>500
500/500
500
62.5/62.5
62.5
>500/>500
>500
>250/>250
>250
0.49/0.49
0.49
7.81/7.81
7.81
7.81/7.81
7.81
Klebsiella pneumoniae J
1436808 (KP-E))
>500/>500
>500
>500/>500
>500
125/125
125
>500/>500
>500
>250/>250
>250
0.98/0.98
0.98
7.81/7.81
7.81
7.81/7.81
7.81
15.62
15.62/15.62
15.62
31.25
250/250
250
Pseudomonas aeruginosa
>500/>500
>500
500/500
500
125/125
125
>500/>500
>500
>250/>250
>250
3.91/3.91
7.81
15.62/31.25
62.5
15.62/31.25
125
CCM 1961 (PA) c
a
Notes:
B12, B14, B16 mean N-benzyl-N,N-dimethyl-N-dodecylammonium bromide, N-benzyl-N,N-dimethyl-N-tetradecylammonium bromide, N-benzyl-N,
N-dimethyl-N-hexadecylammonium bromide, respectively. The preparation [33] and antimicrobial efficacy of B12–16 has been published elsewhere. The antibacterial
activities against Pseudomonas aeruginosa CCM 1961 were determined after 72 h and 120 h of incubation.

Molecules 2015, 20
3688
The sensitivity of G− bacteria (the last four strains) is much lower compared to benzalkonium salts.
The lower potency of compounds 11–13 is evident (higher MIC and MBC). However, an unexpected
sensitivity of PA to compound 12 was observed. With few exceptions, compounds 9, 10, 14 and 15
showed no potency against G− bacteria.
2.3. Cytotoxicity
Cell viability assay confirmed an expected trend that increasing length of carbon chain results in
higher cytotoxicity, probably due to higher lipophilicity of the drug, which facilitates penetration into
the cell. Comparing the cytotoxic potential of individual analogues, none of the new compounds
exceeded its benzalkonium analogue (B12) when it comes to the safety potential (Table 4).
Table 4. Cytotoxic potential of the prepared compounds (9–15) and selected derivatives of
N-benzalkonium (B12–B16). The in vitro activities are expressed as IC50 (μmol/L) ± SEM
(n = 3).
IC50 (μmol/L); 24 h Incubation ± SEM
Cell Line
a
a
a
9
10
11
12
13
14
15
B12
B14
B16
CHO-K1 >1000 108 ± 5
16 ± 2 14 ± 1 7 ± 1 3.5 ± 0.1 2.9 ± 0.2 29 ± 3 24 ± 4 15 ± 1
a
Note:
B12, B14, B16 mean N-benzyl-N,N-dimethyl-N-dodecylammonium bromide, N-benzyl-N,
N-dimethyl-N-tetradecylammonium bromide, N-benzyl-N,N-dimethyl-N-hexadecylammonium bromide,
respectively. The cytotoxic potential of B12–16 has been published elsewhere [33].
3. Experimental Section
3.1. Synthesis
The pyridinium-4-aldoxime salts 10–14 were prepared by reaction of 4-pyridinealdoxime
(1; 8.19 mmol) with 1-bromoalkane (2; 12.46 mmol) in CH3CN (12 mL), as shown in Scheme 1. The
mixture of 4-pyridinealdoxime with 1-bromoalkane in CH3CN was stirred under reflux for 48 h, and
the prepared salts were obtained as white crystals by crystallization from acetone, filtered, washed with
acetone and allowed to dry at room temperature The second method for the synthesis (Scheme 1) of
monoquaternary pyridinium-4-aldoxime salts (3–9) was as follows: Pure 4-pyridinealdoxime (1; 1eq
8.19 mmol) in dry ethanol (30 mL) was mixed with 1-bromoalkane (2; 1,4 eq 12.46 mmol). The
mixture was refluxed for 48 h. The solution was evaporated under reduced pressure and the crude oily
product was recrystallized from ethyl acetate, filtered, washed with ethyl acetate, and allowed to dry at
room temperature.
The progress of the reaction was monitored y TLC (mobile phase ethyl acetate/methanol = 100:1). All
prepared products 9–15 were characterized by 1H-NMR, 13C-NMR, elementary analysis and MS analysis.
Acquired yields (%), melting points (Boetius, uncorrected) and ClogP of prepared salts 9–15 are
summarized in Table 1. All chemicals were reagent or higher grade of purity and were purchased from
Sigma-Aldrich. The progress of the reaction was checked by Thin Layer Chromatography (TLC)
(Merck Milipore Silica gel 60G/UV254, Darmstadt, Germany) with UV detection using wavelength
1
13
254 nm. The H-NMR and C-NMR spectra were recorded with a Varian Mercury-VxBB 300 with
frequencies 300.07 MHz for 1H and 75.46 MHz for 13C. For 1H δ are given in parts per million (ppm)

Molecules 2015, 20
3689
relative to DMSO (δ = 2.50) and for 13C relative to DMSO (δ = 39.43). LogP and ClogP were
calculated with PC software CS ChemBioDraw Ultra 13.0 (CambridgeSoft, Cambridge, MA, USA).
1
4-Hydroxyiminomethyl-1-octylpyridinium bromide (9). H-NMR (300 MHz, DMSO-d6): δ 12.82 (s,
1H, OH); 9.08 (d, J = 6.3 Hz, 2H, 2 × ArH); 8.44 (s, 1H, CH); 8.24 (d, J = 6.7 Hz, 2H, 2 × ArH); 4.57
(t, J = 7.3 Hz, 2H, NCH2-); 1.97–1.81 (m, 2H, CH2); 1.35–1.16 (m, 10H, 5 × CH2); 0.85 (t, J = 6.5 Hz,
13
3H, CH3). C-NMR (75 MHz, DMSO-d6): δ 148.5, 145.2, 145.1, 124.2, 60.4, 49.2, 31.5, 30.7, 28.9,
25.5, 22.3, 14.1. ESI-MS: m/z 235.00 [M+] (counted for: [C14H23N2O]+ 235,18). Anal. Calcd. for
C14H23BrN2O: 53.34% C; 7.35% H; 8.89% N. Found: 54.23% C; 7.31% H; 8.22% N.
1
1-Decyl-4-hydroxyiminomethylpyridinium bromide (10). H-NMR (300 MHz, DMSO-d6): δ 12.80 (s,
1H, OH); 9.08 (d, J = 6.4 Hz, 2H, 2 × ArH); 8.43 (s, 1H, CH); 8.23 (d, J = 6.6 Hz, 2H, 2 × ArH); 4.57
(t, J = 7.3 Hz, 2H, NCH2); 1.96–1.81 (m, 2H, CH2); 1.33–1.17 (m, 14H, 7 × CH2); 0.83 (t,
13
J = 6.6 Hz, 3H, CH3). C-NMR (75 MHz, DMSO-d6): δ 148.5, 145.2, 145.1, 124.2, 60.4, 49.2, 31.5,
30.7, 29.1, 28.9, 25.5, 22.3, 14.1. ESI-MS: m/z 263.00 [M+] (counted for: [C16H27N2O]+ 263,21). Anal.
Calcd. for C16H27BrN2O: 55.98% C; 7.93% H; 8.16% N. Found: 55.38% C; 7.62% H; 8.15% N.
1
1-Dodecyl-4-hydroxyiminomethylpyridinium bromide (11). H-NMR (300 MHz, DMSO-d6): δ 12.80
(s, 1H, OH); 9.09 (d, J = 6.4 Hz, 2H, 2 × ArH); 8.43 (s, 1H, CH); 8.23 (d, J = 6.7 Hz, 2H, 2 × ArH);
4.57 (t, J = 7.3 Hz, 2H, NCH2); 1.97–1.80 (m, 2H, CH2); 1.32–1.16 (m, 18H, 9 × CH2); 0.83 (t,
13
J = 6.7 Hz, 3H, CH3). C-NMR (75 MHz, DMSO-d6): δ 148.5, 145.2, 145.1, 124.2, 60.4, 49.2, 31.5,
30.7, 29.1, 29.0, 28.9, 28.8, 28.6, 25.5, 22.3, 14.2. ESI-MS: m/z 291.00 [M+] (counted for:
[C18H31N2O]+ 291.24). Anal. Calcd. for C18H31BrN2O: 58.22% C; 8.41% H; 7.54% N. Found: 57.70%
C; 8.44% H; 7.54% N.
4-Hydroxyiminomethyl-1-tetradecylpyridinium bromide (12). 1H-NMR (300 MHz, DMSO-d6): δ 12.81
(s, 1H, OH); 9.07 (d, J = 6.4 Hz, 2H, 2 × ArH); 8.43 (s, 1H, CH); 8.23 (d, J = 6.5 Hz, 2H,
2 × ArH); 4.56 (t, J = 7.2 Hz, 2H, NCH2); 1.97–1.81 (m, 2H, CH2); 1.32–1.17 (m, 22H, 11 × CH2);
0.83 (t, J = 6.6 Hz, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6): δ 148.5, 145.3, 145.2, 124.2, 60.2, 49.9,
31.4; 30.7, 30.1, 30.0, 29.8, 29.7, 29.3, 29.1, 28.6, 25.5, 22.3, 14.1. ESI-MS: m/z 319.00 [M+] (counted
for: [C20H35N2O]+ 319.27). Anal. Calcd. for C20H35BrN2O: 60.14% C; 8.33% H; 7.01% N. Found:
59.65% C; 8.98% H; 7.02% N.
1-Hexadecyl-4-hydroxyiminomethylpyridinium bromide (13). 1H-NMR (300 MHz, DMSO-d6): δ 12.82
(s, 1H, OH); 9.07 (d, J = 6.4 Hz, 2H, 2 × ArH); 8.44 (s, 1H, CH); 8.24 (d, J = 6.7 Hz, 2H,
2 × ArH); 4.57 (t, J = 7.2 Hz, 2H, NCH2); 1.96–1.82 (m, 2H, CH2); 1.32–1.18 (m. 26H, 13 × CH2);
0.84 (t, J = 6.4 Hz, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6): δ 148.5, 145.3, 145.2, 124.2, 60.2, 49.9,
31.4; 30.7, 30.1, 30.0, 29.8, 29.7, 29.6, 29.3, 29.1, 28.9, 28.6, 25.5, 22.3, 14.1. ESI-MS: m/z 347.00
[M+] (counted for: [C22H39N2O]+ 347.31). Anal. Calcd. for C22H39BrN2O: 61.81% C; 9.20% H; 6.55% N.
Found: 61.52% C; 9.24% H; 6.64% N.
4-Hydroxyiminomethyl-1-octadecylpyridinium bromide (14). 1H-NMR (300 MHz, DMSO-d6): δ 12.82
(s, 1H, OH); 9.08 (d, J = 6.4 Hz, 2H, 2 × ArH); 8.44 (s, 1H, CH); 8.24 (d, J = 6.6 Hz, 2H, 2 × ArH);

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4.57 (t, J = 7.3 Hz, 2H, NCH2); 1.97–1.81 (m, 2H, CH2); 1.35–1.15 (m, 30H, 15 × CH2); 0.84 (t,
13
J = 6.7 Hz, 3H, CH3). C-NMR (75 MHz, DMSO-d6): δ 148.5, 145.3, 145.2, 124.2, 60.2, 49.9, 31.4;
30.7, 30.1, 30.0, 29.8, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 28.9, 28.6, 25.5, 22.3, 14.1. ESI-MS: m/z
375.34 [M+] (counted for: [C24H43N2O]+ 375.34). Anal. Calcd. for C24H43BrN2O: 63.28% C; 9.51% H;
6.15% N. Found: 62.66% C; 9.67% H; 6.09% N.
1
Eicosyl-4-hydroxyiminomethylpyridinium bromide (15). H-NMR (300 MHz, DMSO-d6): δ 12.82 (s,
1H, OH); 9.05 (d, J = 6.2 Hz, 2H, 2 × ArH); 8.43 (s, 1H, CH); 8.23 (d, J = 6.5 Hz, 2H, 2 × ArH); 4.56
(t, J = 7.3 Hz, 2H, NCH2); 1.98–1.80 (m, 2H, CH2); 1.33–1.17 (m, 34H, 17 × CH2); 0.85 (t, J = 6.6 Hz, 3H,
CH3). 13C-NMR (75 MHz, DMSO-d6): δ 148.5, 145.3, 145.2, 124.2, 60.2, 49.9, 31.4; 30.7, 30.1, 30.0,
29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 28.9, 28.8, 28.6, 25.5, 22.3, 14.1. ESI-MS: m/z 403.50
[M+] (counted for: [C26H47N2O]+ 403.37). Anal. Calcd. for C26H47BrN2O: 64.58% C; 9.80% H; 5.79%
N. Found: 63.94% C; 9.80% H; 5.76% N.
3.2. HPLC Analysis
After the preparation of the whole set of 4-PA salts differing in the length of alkyl chain, we have
developed an appropriate method for their resolution in a mixture using HPLC. The HPLC system
consisted of a P200 gradient pump (Spectra-Physics Analytical, Fremont, CA, USA), a 7125 injection
valve—10 μL loop (Rheodyne, Cotati, CA, USA), a UV1000 detector (Spectra-Physics Analytical,
Fremont, CA, USA) and CSW Chromatography Station 1.5 software (DataApex, Praha, Czech republic).
For analyses a 250 × 4.6 mm I.D. Waters Spherisorb Cyano (5 µm) column was used (Supelco Inc.,
Bellefonte, PA, USA). The mobile phase was 45% acetonitrile and 55% water. This mixture was
prepared as a 0.1 M sodium acetate solution. Finally, the pH was adjusted with acetic acid to 5000. It
was delivered isocratically at a flow-rate of 1 mL/min. The absorbance was measured at 257 nm.
Retention times are summarized in Table 1.
3.3. In-Vitro Antimicrobial Testing
3.3.1. Antifungal Activity
In vitro antifungal activity of the prepared compounds was evaluated on a panel of eight clinical
isolates of fungi, four yeasts (C. albicans ATCC 44859, C. krusei E28, C. tropicalis 156, C. glabrata
20/I) and four filamentous fungi (Trichosporon asahii 1188, Aspergillus fumigatus 231, Absidia
corymbifera 272, Trichophyton mentagrophytes 445). All strains were part of the collection of fungal
strains and are deposited at the Department of Biological and Medical Sciences, Faculty of Pharmacy,
Charles University, Hradec Kralove, Czech Republic. The ATCC strains C. albicans ATCC 90028,
C. parapsilosis ATCC 22019, and C. krusei ATCC 6258 served as the quality control strains.
All the isolates were maintained on Sabouraud dextrose agar prior to being tested. Minimum
inhibitory concentration (MIC) was determined by the modified microdilution format of the CLSI
M27-A3 and M38-A2 for yeasts and filamentous fungi, respectively [42,43]. Dimethyl sulfoxide
(Sigma, Prague, Czech Republic) served as a diluent for all compounds and its final concentration did
not exceed 2%. RPMI 1640 (KlinLab, Prague, Czech Republic) medium supplemented with L-glutamine

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and buffered with 0.165 M morpholinepropanesulfonic acid (Sigma-Aldrich, Prague) to pH 7.0 by 10 M
NaOH was used as a test medium. The wells of the microdilution tray contained 200 μL of the RPMI
1640 medium with two fold serial dilutions of the prepared compounds (500–0.49 μmol/L) and were
inoculated with 10 μL of suspension. The fungal inoculum in RPMI 1640 was prepared to give a final
concentration of 5 × 103 ± 0.2 cfu/mL and 5 × 104 ± 0.5 cfu/mL for yeasts and moulds, respectively.
The trays were incubated at 36 °C ± 1 °C and MIC was read visually and spectrophotometrically (OD
450 nm) for filamentous fungi and yeasts respectively after 24 and 48 h. The MIC values for the
dermatophytic strain (T. mentagrophytes) were determined after 72 h and 120 h. The MICs were
defined as 80% inhibition (IC80) of the growth of control. Minimum fungicidal concentration (MFC)
was established for all compounds tested as the concentration which provided a decrease in the number
of colonies by ≥99.9% after subculturing of a 100 μL aliquot of each well with maximum growth of
20% of control.
3.3.2. Antibacterial Activity
The in vitro antibacterial activity of the prepared compounds was tested on a panel of eight bacterial
strains (Staphylococcus aureus CCM 4516/08, S. aureus MRSA H 5996/08, S. epidermidis
HK6966/08, Enterococcus sp. HK14365/08, Escherichia coli CCM 4517, Klebsiella pneumoniae D
11750/08, K. pneumoniae J 14368/08, and Pseudomonas aeruginosa CCM 1961). The ATCC strains
also served as the quality control strains; the rest of them were clinical isolates from the patients and
are deposited at the Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles
University, Hradec Kralove, Czech Republic. Before testing the strains were passaged on Mueller-
Hinton Agar (HiMedia, Cadersky-Envitek, Prague, Czech Republic).
Minimum inhibitory concentration (MIC) of the prepared compounds was determined by the
microdilution broth method modified according to standard M07-A07 [44]. Mueller-Hinton Broth
(MH, HiMedia, Cadersky-Envitek, Prague, Czech Republic) adjusted to pH 7.4 (±0.2) was used as the
test medium. DMSO served as a diluent for all compounds and its final concentration did not exceed
2% in the test medium. The wells of the microdilution tray contained 200 μL of the MH broth with
twofold serial dilutions of the compounds (500–0.49 μmol/L) and were inoculated with 10 μL of
bacterial suspension. A bacterial inoculum in sterile water was prepared to match 0.5 McFarland scale
(1.5 × 108 CFU/mL). The MIC values were read visually after 24 h and 48 h incubation at 36 °C ± 1 °C;
for Pseudomonas aeruginosa CCM 1961, the MIC was determined after 72 h and 120 h of incubation.
The MIC was defined as complete inhibition of growth. Minimum bactericidal concentration (MBC)
was established for all compounds tested as the concentration that provided a decrease in the number
of colonies by ≥99.9% after subculturing of a 100 μL aliquot of each well without visible growth.
3.4. Cytotoxicity
Standard MTT assay (Sigma Aldrich, Prague, Czech Republic) was used according to the
manufacturer’s protocol on the CHO-K1 cell-line (Chinese hamster ovary, ECACC, Salisbury, UK) in
order to compare the cytotoxic effect of the studied compounds. The cells were cultured according to
ECACC recommended conditions and seeded at a density of 8000 per well. Briefly, the tested
compounds were dissolved in DMSO and subsequently in the growth medium (F-12) supplemented

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with 10% FBS and 1% penicillin/streptomycin so that the final concentration of DMSO did not exceed
0.5% (v/v). Cells were exposed to the tested compounds for 24 h. The medium was then replaced by a
medium containing 10 μM of MTT and the cells were allowed to produce formazan for another 3 h
under surveillance. Thereafter, the medium with MTT was removed and crystals of formazan were
dissolved in DMSO (100 μL). Cell viability was assessed spectrophotometrically by the amount of
formazan produced. Absorbance was measured at 570 nm with 650 nm reference wavelength on
Synergy HT (BioTek, Winooski, VT, USA). IC50 was then calculated from the control-subtracted
triplicates using non-linear regression (four parameters) of GraphPad Prism 5 software. The final IC50
and SEM value was obtained as the mean of three independent measurements.
4. Conclusions
The whole set of pyridine-4-aldoxime based quaternary ammonium salts with differing length of
alkyl side chain was synthesized (9–15). We have described two possible conditions for the
preparation. Both methods are usable for the preparation and no significant difference in the yields was
observed. The compounds were analyzed by NMR, EA and MS analysis and the logP was calculated
for each compound. Furthermore, an HPLC experimental protocol was found that was fully applicable
for purity evaluation and could be easily used for characterization of mixtures of all homologues.
Antimicrobial (antifungal and antibacterial) activity evaluation confirmed that compounds having an
alkyl chain of C12–C16 outperformed the rest of the tested compounds. Yeast-type fungi were the most
sensitive towards those analogues whereas the C16 analogue was completely ineffective against
filamentous fungi. Antibacterial assessment revealed versatility of C14 and relatively high efficacy of
C16 against G+ strains and C14 against G− strains. Notably, none of the studied compounds exceeded
the efficacy and versatility of the benzalkonium C12 analogue, and benzalkonium analogues also
showed a lower effect in the cell viability assay.
Acknowledgments
The publication is co-financed by the European Social Fund and the state budget of the Czech
Republic. Post-doctoral Project no. CZ.1.07/2.3.00/30.0061, CZ.1.07/2.3.00/30.0016. This work was
also supported by long development plan 1011 and MH CZ—DRO (UHHK, 00179906). We wish to
thank Jiri Kunes (Faculty of Pharmacy, Charles University in Prague, Hradec Kralove) for
measurement of NMR spectra.
Author Contributions
J.M. and D.M. were responsible for the preparation of the compounds, identification and purity
analysis. J.M. also ensured biological testing on microbial strains and manuscript preparation.
R.D. performed HPLC analysis. O.S. and M.P. were responsible for the cell viability testing and
structure-activity relationship evaluation. M.D. and K.K. participated on the design of new
compounds, management of the study, were responsible for the funding, final correction of the
manuscript and correspondence.

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Conflicts of Interest
The authors declare no conflict of interest. The founding sponsors had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).