Wide-Antimicrobial Spectrum of Picolinium Salts

Article abstract of DOI:10.3390/molecules25092254

Nosocomial infections, which greatly increase morbidity among hospitalized patients, together with growing antibiotic resistance still encourage many researchers to search for novel antimicrobial compounds. Picolinium salts with different lengths of alkyl chains (C₁₂, C₁₄, C₁₆) were prepared by Menshutkin-like reaction and evaluated with respect to their biological activity, i.e., lipophilicity and critical micellar concentration. Picolinium salts with C₁₄ and C₁₆ side chains achieved similar or even better results when in terms of antimicrobial efficacy than benzalkoniums; notably, their fungicidal efficiency was substantially more potent. The position of the methyl substituent on the aromatic ring does not seem to affect antimicrobial activity, in contrast to the effect of length of the N-alkyl chain. Concurrently, picolinium salts exhibited satisfactory low cytotoxicity against mammalian cells, i.e., lower than that of benzalkonium compounds, which are considered as safe.

Full text of DOI:10.3390/molecules25092254

molecules
Article
Wide-Antimicrobial Spectrum of Picolinium Salts
Sarka Salajkova 1,2, Marketa Benkova 1,3 , Jan Marek 1,3, Radek Sleha 3, Lukas Prchal 1,
David Malinak 1,4, Rafael Dolezal 1,4 , Kristina Sepcˇic´
, Nina Gunde-Cimerman 5
,
5
Kamil Kuca 1,4,
*
and Ondrej Soukup 1,
*
1
Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove,
Czech Republic; sarka.salajkova@img.cas.cz (Sa.S.); Marketa.Benkova@fnhk.cz (M.B.);
jan.marek@fnhk.cz (J.M.); lukas.prchal@fnhk.cz (L.P.); david.malinak@uhk.cz (D.M.);
rafael.dolezal@uhk.cz (R.D.)
2
3
4
5
Department of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences,
Videnska 1083, 142 20 Prague, Czech Republic
Department of Epidemiology, University of Defence in Brno, Trebesska 1575, 500 05 Hradec Kralove,
Czech Republic; radek.sleha@unob.cz
Department of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62,
500 03 Hradec Kralove, Czech Republic
Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana,
Slovenia; Kristina.Sepcic@bf.uni-lj.si (K.S.); Nina.Gunde-Cimerman@bf.uni-lj.si (N.G.-C.)
Correspondence: kamil.kuca@uhk.cz (K.K.); ondrej.soukup@fnhk.cz (O.S.);
Tel.: +420-493-332-509 (K.K.); +420-495-833-447 (O.S.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editors: Cheng-Wei Tom Chang, Jon Y. Takemoto and Jixun Zhan
Received: 17 April 2020; Accepted: 8 May 2020; Published: 11 May 2020
Abstract: Nosocomial infections, which greatly increase morbidity among hospitalized patients,
together with growing antibiotic resistance still encourage many researchers to search for novel
antimicrobial compounds. Picolinium salts with different lengths of alkyl chains (C12, C14, C16) were
prepared by Menshutkin-like reaction and evaluated with respect to their biological activity, i.e.,
lipophilicity and critical micellar concentration. Picolinium salts with C14 and C16 side chains achieved
similar or even better results when in terms of antimicrobial efficacy than benzalkoniums; notably,
their fungicidal efficiency was substantially more potent. The position of the methyl substituent on
the aromatic ring does not seem to affect antimicrobial activity, in contrast to the effect of length
of the N-alkyl chain. Concurrently, picolinium salts exhibited satisfactory low cytotoxicity against
mammalian cells, i.e., lower than that of benzalkonium compounds, which are considered as safe.
Keywords: picolinium salts; quaternary ammonium compounds; surfactant; critical micellar
concentration; antimicrobial activity; cytotoxicity
1. Introduction
Surfactants based on quaternary ammonium salts (QASs) are widely used from academic
establishments to industry [
used as emulsifiers, moisturizers [
hand cleaning agents and topical hand sanitizers [
1
–
4
]. Because of their surface-active properties [
] and cleaning agents, as well as ointment disinfectants, pre-operative
]. QASs can also be utilized as potential hydrolytic
5–7], they are commonly
8
9
micellar catalysts to accelerate chemical decomposition, and such compounds can find uses as
decontaminants [10–13] or in the preparation and coating of nanoparticles [14].
Most importantly, QASs have been used as disinfectants for years. The correct practice of
disinfectants or their cocktail use within hospitals has long been considered the most appropriate
first-line defense for decreasing the incidence of nosocomial infections and minimizing the prescription
Molecules 2020, 25, 2254; doi:10.3390/molecules25092254
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2254
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of antibiotics [
1,
15–
19]. The ordinarily applied QASs are derivatives of benzalkonium, benzoxonium,
pyridinium or cetrimonium salts [11].
One of the most beneficial features of QASs is their cytotoxic effect at very low concentration
to a broad spectrum of microorganisms, including bacteria, fungi, parasites and enveloped viruses,
while showing relatively low toxicity to eukaryotic cells [20,21]. QASs generally act by disruption of
the cytosolic membrane and outer lipid bilayer via replacement of bivalent ions and attachment to
the membrane, followed by the incorporation of the hydrophobic side chain into the phospholipid
bilayer and the creation of a mixed micelle with the phospholipids, resulting ultimately in cytosol
leakage (Figure 1A) [10
negatively charged phospholipids, eukaryotic membranes mostly contain zwitterionic lipids and
cholesterol [23 25]. Such a difference in composition is a good precondition for a selective effect
against microbes, and therefore, QAS should exhibit selectivity to microorganisms over eukaryotic
cells [ 15 25]. Indeed, selectivity is obvious even among microorganisms. Gram-positive (G+) bacteria
,18,22]. Whereas bacterial membranes are composed predominantly of
–
4,
,
are more sensitive than Gram-negative (G-). The increased resistance of G- is most likely a consequence
of the presence of the outer membrane layer which grants the cells stronger physicochemical protection
(Figure 1B) [18
activity is nonlinear. Optimal bioactivity is exhibited by C12–C14 chains for G+, and C14–C16 chains for
G- bacteria [ 15 18 26]. However, the lipophilicity of the side chains seems to be a crucial factor [ 27],
,25]. Interestingly, the relationship between the alkyl side-chain length and antibacterial
5,
,
,
4,
although the precise mechanism of action and structure–activity relationship is not yet fully understood.
Figure 1. Cidal effect of QAS. Disintegration mechanism of the membrane by QAS attachment on to
the membrane, followed by incorporation of side chains into the membrane resulting in membrane
rupture and cytosol leakage (A). Fundamental composition of cell walls (B).

Molecules 2020, 25, 2254
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Picolinium salts are pyridinium derivatives and can be prepared by modification of the Menshutkin
reaction commonly used for the preparation of QAS [18 26 28 31]. Picolinium salts have, in many
cases, been used as intermediates for the preparation of donor-acceptor molecules for the construction
of promising photo- and electro-active materials [29 32 38]. In addition, their typical physicochemical
features such as surface activity and micellization have been intensively studied in the line of their
possible application [39 44]. For instance, N-(n-hexadecyl)- and N-(n-heptadecyl)-3-methylpyridinium
,
, –
,
–
–
bromide or 1-methyl-3-octylpyridinium alkyl sulfate were found to be effective for micelle-based
drug delivery to improve drug solubility and the bioavailability of anionic drugs and metal
complexes [28
as promising antagonists of neuronal nicotinic acetylcholine receptors, mediating nicotine-evoked
dopamine release [45 49]. Although the antimicrobial activities of quaternary ammonium salts are
,43]. The Crooks’ group systematically investigated mono- and bis-picolinium derivatives
–
generally known, the published results regarding the antimicrobial activities of picolinium salts
are inconsistent. Several publications have reported an effect of some picolinium representatives,
such as 1-dodecyl-4-methylpyridinium bromide [42], 1-dodecyl-3-methylpyridinium bromide [30],
3-methyl-1-tetradecylpyridinium bromide and 1-hexadecyl-3-methylpyridinium bromide [43],
against Micrococcus luteus [30], Staphylococcus aureus [30,31,42,43], Enterobacter aerogenes [30],
Escherichia coli [30,31,42,43], Klebsiella pneumonia [43] and Pseudomonas æruginosa [42]. However, there
has been no study systematically evaluating the antimicrobial effectiveness of ortho, meta and para
N-alkylpicolinium salts differing in their alkyl chain length. Furthermore, wide-spectrum efficacy
against yeasts, fungi and viruses is unknown.
Therefore, the aim of this study is to systematically describe the biological properties of a
picolinium series on a wide set of microorganisms, including bacteria, fungi and viruses. Besides
evaluating the basic physicochemical parameters, we focused on the antimicrobial action in an attempt
to elucidate the structure-activity relationship against various microbes and against mammalian cells
for selectivity reasons.
2. Results and Discussion
2.1. Synthesis and Physicochemical Properties of Picolinium Salts
Surfactants 4a
were chosen to facilitate the bimolecular nucleophilic substitution of the corresponding bromoalkane
with the appropriate methylpyridine isomers in acetonitrile (ACN) (Scheme 1) [18]. The crude
–6c were prepared in one step by Menshutkin-like reaction. The reaction conditions
1
–3
a–c
products were crystallized from diethyl ether to afford 4–6 in moderate to high yields (Table 1).
Lipophilicity is an important molecular descriptor governing the intercalation of the QAS side
chain into the cell membrane, with its subsequent disruption and cell death. The lipophilicity of the
compounds was estimated by chromatographic study using a simple HPLC-MS method with isocratic
elution, as described previously [18
,26]. The expected linear relationship between the prolongation
of the alkyl chain of QAS 4a 6c and the increasing lipophilicity driven by the length of the alkyl
–
chain was observed with a high correlation coefficient (R2 < 0.97; values are listed in Figure 2).
The position of the methyl group on the aromatic ring slightly affects the lipophilicity of the molecules.
The position of the methyl group in the para- or meta- position did not result in any change on cLog p;
however, a noticeable effect was observed by introucing this group into the ortho- position. Thus, the
lipophilicity of the picolinium compounds increased in the order o-picolinium (6a–c) < m-picolinium
(5a–c) = p-picolinium (4a–c).
Scheme 1. Preparation of alkyl chain compounds; n = 5, 6, 7; R = CH3.

Molecules 2020, 25, 2254
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Table 1. Prepared compounds with alkyl chains and their physicochemical properties.
Chain
Length
Yield
[%]
CMC e
[µmol/L]
m.p.b [◦C]
Clog p d
Structure
Cpd a
kc
log k
4a
5a
C12H25
C14H29
50
95
121.9–123.9
125.7–127.4
0.05 ± 0.01
0.35 ± 0.01
−1.28
−0.45
2.21
3.10
14,010
2330
600
6a
C16H33
98
126.8–128.8
1.09 ± 0.01
0.04
3.98
n = 5, 6, 7
n = 5, 6, 7
4b
5b
C12H25
C14H29
80
95
34.0–36.0
72.3–74.3
0.07 ± 0.01
0.37 ± 0.01
−1.14
−0.43
2.21
3.10
9490
2320
860
6b
C16H33
96
70.8–72.6
1.18 ± 0.01
0.07
3.98
4c
5c
C12H25
C14H29
88
4
41.4–42.8
60.4–62.3
0.08 ± 0.01
0.36 ± 0.02
−1.12
−0.44
1.36
2.25
14,160
3460
650
6c
C16H33
80
79.9–81.9
1.10 ± 0.01
0.04
3.14
n = 5, 6, 7
a
Compound (Cpd), b Melting point (m.p.), c Capacity factor (k) was determined by an isocratic LC-MS method,
Marvin was used for drawing, displaying and characterizing chemical structures (Clog p), substructures and
d
reactions, Marvin 17.21.0, ChemAxon (https://www.chemaxon.com), e Critical micellar concentration (CMP).
Figure 2. Correlation between compound structure and physicochemical properties. Graphs show the
lipophilicity of the prepared compounds and their dependency on chain elongation (A). Stauff-Klevens
plots of log CMC and extended chain of surfactants (B).
A specific characteristic of surfactants is their ability to create micelles characterized as the critical
micelle concentration (CMC) [ 11 50 51]. CMC was measured using a conductometric method, and the
parameters of the experiment were set according to our previous investigations [ 26]. Elongation
4,
,
,
9,
of the alkyl chains decreases the CMC value according to the Traube rule (Figure 2). Furthermore,
the position of the methyl group in the hydrophilic head of the compounds has a negligible influence
on micelle formation, which is clear proof that the tail length is the dominant factor for micelle
creation. Pernak et al. demonstrated a relationship between the CMC and the antibacterial properties
of 1-alkyl-3-alkylthiomethylimidazolium chlorides [52,53]. The published results imply that the micelle
formation enhances the local concentration of surfactant with its cationic charge, leading to a stronger
interaction with the negatively charged net of the cell membrane [54,55].
However, it is still not clear how micelle formation affects the antimicrobial properties, and the
published results are inconsistent in this context. The biological activity seems to be not affected
directly by the structure of substituents attached to the polar heads, but rather, by the morphology
of aggregates, such as the size, which is enhanced with increasing the alkyl chain length [56]. In line
with this knowledge, compounds 4a–6c were used in the subsequent biological evaluation far below
their CMC.

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2.2. Antimicrobial Activity of Picolinium Salts
The prepared picolinium salts showed strong antimicrobial properties below their CMC (Figure 3),
as well as a broad spectrum of activities (Figure S2) against chosen microorganisms, including
bacteria, fungi and viruses, while having simultaneously satisfactory cytotoxicity for mammalian
cells. The results were compared with the activity of benzalkonium salts, which are considered the
gold standard of quarternary ammonium salt-based disinfectants (data are shown in Supplementary
Information, SI).
Figure 3. The biological assessment of prepared picolinium salts 4a–6c
concentration (MIC) after 24 h or 48 h incubation against Gram-positive ( or
bacteria ( or ). Minimum bactericidal concentration (MBC) after 48 h treatment against Gram-positive
) and Gram-negative bacteria ( ). Minimum inhibitory concentrations (MIC) determined on a panel
of four yeasts and three filamentous fungi after 72 h (yeasts)/168 h (filamentous fungi) incubation ( ).
.
Minimum inhibitory
A
C) and Gram-negative
B
D
(
E
F
G
Virucidal efficacy against varicella zoster virus (VZV); 5 min exposure to 0.01%; or 0.005%; expressed as
∆TCID50 (H). Complete data are listed in Tables S1, S2 and S3.

Molecules 2020, 25, 2254
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The antibacterial properties were evaluated as the minimum inhibitory and bactericidal
concentrations (MIC and MBC, respectively) for nine bacterial strains, namely the three G+ strains,
Staphylococcus aureus C1947 (SA), methicillin-resistant Staphylococcus aureus C1926 (MRSA), and
vancomycin-resistant enterococci S2484 (En), and the six G- strains, Acinetobacter baumannii J3474 (AB),
Escherichia coli A1235 (EC), Klebsiella pneumoniae C1950 (BL), extended-spectrum β-lactamase-producing
Klebsiella pneumoniae C1934 (ESBL), Stenotrophomonas maltophilia J3552 (SM), and Yersinia bercovieri
CNCTC6230 (YB). The quaternary ammonium salts with C12 or C14 alkyl chain lengths are considered
the most efficacious against G+ and G bacteria, respectively. Interestingly, the antibacterial effect
−
against each strain, as well as the width of the antibacterial spectrum, was enhanced with increasing
the length of the side chains C12 < C14 < C16. Picolinium salts have a similar trend of activity towards
both G and G+ bacteria (Figure 3A–D), yet the level of efficacy on those bacteria types is considerably
−
different. The MIC and MBC for individual bacterial strains are listed in Table S1 (in SI).
Picolinium salts with a C12 side-chain showed lower activity, particularly against enterococci.
Both C14 and C16 derivatives (5a–6b) exhibited very good efficacy against the tested G+ strains, with
comparable or better antibacterial activity than that of benzalkonium salts (5B, 6B) (Figure S2). Stronger
activities than those of benzalkonium salts were shown against Staphylococcus aureus by compounds
with a C14 or C16 side chain. In the case of MRSA, the activities of compounds 5a–6b were similar to
those of benzalkonium salts. The strains of G+ bacteria were sensitive to the test compounds in the
order: SA > MRSA > En.
The antibacterial activities against G
bacteria to antimicrobial agents presumably arises from the higher lipid content of the cell membrane and
the presence of the outer phospholipid membrane layer with proteoglycans, which serves as a stronger
−
bacteria were significantly lower. The resistance of G
−
physicochemical barrier [18
,25,54,55,57]. The most promising activity was exhibited by compounds
with a C16 alkyl chain, followed by those with C14, which attained similar activity as the standards (5B
,
6B). These analogues also have broad-spectrum efficacy, like benzalkonium salts (see SI). C12-derivatives
had lower antibacterial effects, and poor activity in the case of Stenotrophomonas maltophilia and both
Klebsiella strains. Overall, varying the position of the methyl group on the aromatic ring had a
negligible effect on antibacterial efficacy compared to the elongation of the alkyl chain. Our results
correspond to published data by Viscari et al.; those authors compared the antibacterial efficiency
of 1-dodecyl-4-methylpyridinium bromide (4a) and N-benzyl-N,N-dimethyl-N-dodecylammonium
bromide (4B) as standards against Staphylococcus aureus, Escherichia coli and Pseudomonas æruginosa [42].
The antifungal efficiencies were assessed as the minimum inhibitory concentrations (MIC) for seven
fungi: four yeast strains, i.e., Candida parapsilosis sensu stricto EXF-8411 (CAPA), Rhodotorula mucilaginosa
EXF-8417 (RHMU), Exophiala dermatitidis EXF-8470 (EXDE), Aureobasidium melanogenum EXF-8432
(AUME); and the three filamentous fungi, i.e., Bisifusarium dimerum EXF-8427 (BIDI), Penicillium
chrysogenum EXF-1818 (PECH) and Aspergillus versicolor EXF-8692 (ASVE).
A similar relationship was observed between antifungal activity and picolinium side-chain
prolongation as for antibacterial activity, i.e., the increasing trend of efficiency was with elongation
of the side-chain C12 < C14 < C16. All prepared QASs had moderate to very good efficacy against
Aureobasidium melanogenum, Rhodotorula mucilaginosa and Exophiala dermatitidis, while the remaining
tested fungi showed only minor sensitivity to compounds bearing a C12 chain (4a
–c). Picolinium salts
5a 6c were efficient against a whole spectrum of selected fungi, even more so than benzalkonium salts.
–
Again, the position of the methyl substituent on the aromatic ring does not seem to affect antifungal
activity significantly compared to the effect of the alkyl chain length. MIC for individual fungal strains
are listed in Table S2 (in SI).
Finally, the antiviral activity against varicella zoster virus (VZV) was determined in order to
evaluate the width of the antimicrobial spectrum. The reduction factors, expressed as the change of
values of 50% tissue culture infectious doses (logTCID50) of viral titer before and after 5 min exposure
time to compounds being tested are summarized in Table S3 (see SI). The efficacy of the antiviral
activity of antiviral drugs should exhibit at least a reduction factor of 4 in the virus titer. However, none

Molecules 2020, 25, 2254
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of our compounds or standards achieved such a result. The highest effect against VZV was manifested
by benzalkonium salts with C12 alkyl chain (reduction factor = 4.67), and from the picolinium series 5a
and 5b, which exerted some activity at a concentration of 0.01% (reduction factor = 1.34). Predictably,
higher antiviral activity could be expected at higher concentrations, i.e., typically those used in practice
in the case of QASs, against enveloped viruses. Unfortunately, no SAR can be deduced, since C16
analogues, due to their limited solubility, were tested at half the concentration (0.005%) of C12 and C14
analogues (0.01%).
2.3. Cytotoxic Effect on Mammalian Cells
A cell viability assay (the MTT assay) of the prepared compounds was performed on eukaryotic
mammalian hamster ovarian cells (CHO-K1), and is expressed as IC50. In contrast to the cidal
effects of the compounds, their cytotoxic potential on eukaryotic cells was linearly dependent on
their lipophilicity. Figure 4 shows a significant trend that side chain elongation relates to increasing
lipophilicity of the compound and higher cytotoxic effect, facilitated by better penetration into the cell
membrane. The influence of the substituent position on the aromatic ring was more pronounced in
comparison to the cidal effects, with cytotoxicity increasing in the order o-picolinium < p-picolinium <
m-picolinium. The cytotoxicity, expressed as the IC50 for individual compounds, is listed in Table S4
(in SI).
Figure 4. Cytotoxicity of prepared QASs 4a–6c (A) and their comparison with benzalkonium salts
4–6B (B).
The comparison of picolinium and benzalkonium salts with 3-hydroxypiridinium [18] clearly
proved that the introduction of lipophobic substituents on pyridine contributes to the overall
lipophilicity of compounds and increases their cytotoxic potential (Figure 4B). Moreover, the
introduction of the hydroxyl substituent resulted in higher hydrophilicity of the whole structure and
reduced cytotoxicity.
In summary, the cytotoxicity is clearly based on the lipophilicity of the compound in the way that
the increasing lipophilicity causes higher toxic potential. However, the alkyl chain showed the major
impact of the overall lipophilicity of the compounds; the minor contribution of the methyl substituent
on aromatic ring was observed in the case of compounds 4a–6c. Nevertheless, picolinium salts show a
similar effect on cell viability as the benzalkoniums, which are considered to be nontoxic surfactants.
3. Materials and Methods
3.1. General Synthetic Data
All commercial reagents for synthesis were purchased as reagent grade from Sigma-Aldrich
(Prague, Czech Republic) and used without further purification. Anhydrous solvents for synthesis
were purchased from Sigma-Aldrich (Czech Republic), and the others were purchased from Penta
chemicals Co or VWR International (Prague, Czech Republic). Acetonitrile, methanol and formic
acid for LC-MS analyses were obtained from Sigma-Aldrich in LC-MS grade purity (Czech Republic).

Molecules 2020, 25, 2254
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Ultrapure water was produced by Barnstead Smart2Pure 3 UV/UF apparatus (ThermoFisher Scientific,
Bremen, Germany). Thin-layer chromatography was carried out on Merck silica gel 60 F254 analytical
plates (Prague, Czech Republic); detection was accomplished with phosphomolybdic acid stain (10 g
PMA in 100 mL EtOH), or ultraviolet light (254 nm lamp). Uncorrected melting points of prepared
compounds were determined by Melting Point Apparatus—Stuart SMP30 (Melting Point Apparatus –
Stuart SMP30, Eaton, United Kingdom).
1H NMR and 13C NMR spectra of the prepared compounds were recorded at ambient temperature
on a Varian S500 spectrometer (499.87 MHz for 1H and 125.71 MHz for 13C, Varian, Palo Alto, CA,
USA). The NMR spectra were processed with MestReNova 12.0.4-22023, 2018 Mestrelab Research S.L.
The chemical shift values for 1H and 13C NMR spectra are reported in ppm (
solvent peak DMSO (δH = 2.55 ppm, δC = 39.52 ppm). For 1H,
δ
) relative to residual
δ
are given in parts per million (ppm)
relative to solvent, and the coupling constants (J) are expressed in Hertz (Hz). Signals are quoted as
s = singlet, d = doublet, t = triplet or m = multiplet.
The HPLC-MS analysis of intermediates and final products was performed using a Dionex UltiMate
3000 analytical system coupled with a Q Exactive Plus hybrid quadrupole-orbitrap spectrometer
(ThermoFisher Scientific, Bremen, Germany) according to a gradient method using a Waters Atlantis
dC18 (2.1
×
100 mm/3 µm; Waters, Ireland) column previously described by Benkova et al. (2019) [26].
The purity of samples was determined from UV chromatograms recorded at a wavelength of 254 nm,
and the HRMS results were determined from the total ion current spectra.
3.1.1. General Procedure for the Synthesis of Quaternary Ammonium Salts.
Appropriate bromide analogues (0.25 mmol, 1 eq) were dissolved in ACN (0.6 mL), and the
corresponding amine (1.2 eq) was added. The reaction mixture was stirred under reflux for 48 h, and the
solvent was evaporated under reduced pressure. The crude products were purified by crystallization
from diethyl ether, filtered, washed with diethyl ether at ambient temperature, and allowed to dry to
obtain corresponding products in yields 50–98%.
1-Dodecyl-4-methylpyridinium bromide (4a). Compound 4a was obtained as white crystals (50%); m.p.
41.4 ◦C–42.8 ◦C. 1H-NMR (500 MHz, DMSO)
δ
(ppm): 8.99 (d, J = 6.6 Hz, 2H), 8.00 (d, J = 6.5 Hz, 2H),
4.55 (t, J = 7.4 Hz, 2H), 2.60 (s, 3 H), 1.92–1.83 (m, 2H), 1.30–1.17 (m, 18 H), 0.84 (t, J = 6.9 Hz, 3H).
13C-NMR (126 MHz, DMSO)
δ (ppm): 158.69, 143.69, 128.30, 59.80, 31.26, 30.57, 28.97, 28.88, 28.76,
28.67, 28.35, 25.34, 22.06, 21.33, 13.92. HRMS (HESI+): calculated for [M+] C18H32N+ (m/z) 262.2529;
found 262.2527.
4-Methyl-1-tetradecylpyridinium bromide (5a). Compound 5a was obtained as white crystals (95%); m.p.
60.4 ◦C–62.3 ◦C. 1H-NMR (500 MHz, DMSO)
δ
(ppm): 8.96 (d, J = 6.7 Hz, 2H), 7.99 (d, J = 6.6 Hz,
2H), 4.53 (t, J = 7.4 Hz, 2H), 2.60 (s, 3H), 1.91–1.80 (m, 2H), 1.36–1.09 (m, 22H), 0.84 (t, J = 6.9 Hz, 3H).
13C-NMR (126 MHz, DMSO)
δ (ppm): 158.71, 143.68, 128.31, 59.84, 31.27, 30.56, 29.03, 29.01, 28.99,
28.97, 28.89, 28.77, 28.68, 28.35, 25.35, 22.07, 21.34, 13.93. HRMS (HESI+): calculated for [M+] C20H36N+
(m/z) 290.2842; found: 290.2837.
1-Hexadecyl-4-methylpyridinium bromide (6a). Compound 6a was obtained as white crystals (98%); m.p.
79.9 ◦C–81.9 ◦C. 1H-NMR (500 MHz, DMSO)
δ
(ppm): 8.98 (d, J = 6.7 Hz, 2H), 8.00 (d, J = 6.6 Hz,
2H), 4.54 (t, J = 7.4 Hz, 2H), 2.61 (s, 3H), 1.95–1.78 (m, 2H), 1.44–1.13 (m, 26H), 0.84 (t, J = 6.9 Hz, 3H).
13C-NMR (126 MHz, DMSO)
δ (ppm): 158.69, 143.68, 128.30, 59.81, 31.26, 30.56, 29.02, 28.97, 28.89,
28.77, 28.67, 28.36, 25.34, 22.06, 21.32, 13.91. HRMS (HESI+): calculated for [M+] C22H40N+ (m/z):
318.3155; found: 318.3149.
1-Dodecyl-3-methylpyridinium bromide (4b). Compound 4b was obtained as white crystals (80%); m.p.
34.0 ◦C–36.0 ◦C. 1H-NMR (500 MHz, DMSO)
δ
(ppm): 9.09 (s, 1H), 8.98 (d, J = 6.0 Hz, 1H), 8.46 (d,
J = 8.0 Hz, 1H), 8.10–8.04 (m, 1H), 4.60–4.54 (m, 2H), 2.50 (s, 3H), 1.96–1.87 (m, 2H), 1.48–1.14 (m, 18H),
0.84 (t, J = 6.9 Hz, 3H); 13C-NMR (126 MHz, DMSO)
(ppm): 145.69, 144.22, 141.94, 138.68, 127.29,
δ

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60.49, 31.26, 30.62, 28.97, 28.89, 28.75, 28.67, 28.36, 25.37, 22.06, 17.83, 13.92. HRMS (HESI+): calculated
for [M]+ C18H32N+ (m/z): 262.2529; found: 262.2525.
3-Methyl-1-tetradecylpyridinium bromide (5b). Compound 5b was obtained as white crystals (95%); m.p.
72.3 ◦C–74.3 ◦C. 1H-NMR (500 MHz, DMSO)
δ
(ppm): 9.08 (s. 1H), 8.97 (d, J = 6.0 Hz, 1H), 8.46 (d,
J = 8.0 Hz, 1H), 8.09–8.04 (m, 1H), 4.59–4.54 (m, 2H), 2.50 (s, 3H), 1.95–1.87 (m, 2H), 1.55–0.97 (m, 22H),
0.85 (t, J = 6.9 Hz, 3H); 13C-NMR (126 MHz, DMSO)
(ppm): 145.70, 144.21, 141.94, 138.70, 127.29,
δ
60.52, 31.27, 30.62, 29.03, 29.01, 28.99, 28.98, 28.90, 28.75, 28.68, 28.37, 25.38, 22.07, 17.84, 13.92. HRMS
(HESI+): calculated for [M]+ (m/z) C20H36N+ 290.2842; found: 290.2835.
1-Hexadecyl-3-methylpyridinium bromide (6b
70.8 ◦C–72.6 ◦C. 1H-NMR (500 MHz, DMSO)
J = 8.0 Hz, 1H), 8.10–8.03 (m, 1H), 4.60–4.53 (m, 2H), 2.50 (s, 3H), 1.96–1.86 (m, 2H), 1.48–1.14 (m, 26H),
).
Compound 6b was obtained as white crystals (96%); m.p.
δ
(ppm): 9.09 (s, 1H), 8.98 (d, J = 6.0 Hz, 1H), 8.46 (d,
0.84 (t, J = 6.9 Hz, 3H). 13C-NMR (126 MHz, DMSO)
δ (ppm): 145.69, 144.21, 141.94, 138.68, 127.28,
60.50, 31.26, 30.62, 29.02, 28.97, 28.90, 28.75, 28.67, 28.37, 25.38, 22.06, 17.82, 13.91. HRMS (HESI+):
calculated for [M+] C22H40N+ (m/z): 318.31553; found: 318.3149.
1-Dodecyl-2-methylpyridinium bromide (4c). Compound 4c was obtained as white crystals (88%); m.p.
121.9 ◦C–123.9 ◦C. 1H-NMR (500 MHz, DMSO)
δ
(ppm): 9.07 (d, J = 6.7 Hz, 1H), 8.51–8.45 (m, 1H), 8.07
(d, J = 7.8 Hz, 1H), 8.01–7.95 (m, 1H), 4.59–4.54 (m, 2H), 2.85 (s, 3H), 1.88 – 1.79 (m, 2H), 1.42–1.13 (m,
18H), 0.84 (t, J = 6.9 Hz, 3H); 13C-NMR (126 MHz, DMSO)
δ (ppm): 155.04, 145.33, 145.00, 129.92, 125.52,
57.19, 31.25, 29.36, 28.97, 28.96, 28.90, 28.80, 28.67, 28.43, 25.63, 22.05, 19.55, 13.91. HRMS (HESI+):
calculated for [M]+ C18H32N+ (m/z): 262.25293; found: 262.2525.
2-Methyl-1-tetradecylpyridinium bromide (5c). Compound 5c was obtained as white crystals (84%); m.p.
125.7 ◦C–127.4 ◦C. 1H-NMR (500 MHz, DMSO)
δ
(ppm): 9.06 (d, J = 6.0 Hz, 1H), 8.51–8.45 (m, 1H),
8.07 (d, J = 7.8 Hz, 1H), 8.01–7.95 (m, 1H), 4.59–4.52 (m, 2H), 2.85 (s, 3H), 1.88–1.77 (m, 2H), 1.44–1.04
(m, 22H), 0.84 (t, J = 6.9 Hz, 3H); 13C-NMR (126 MHz, DMSO)
(ppm): 155.04 145.33, 145.00, 129.92,
δ
125.52, 57.19, 31.25, 29.36, 29.02, 29.00, 28.97, 28.91, 28.81, 28.67, 28.43, 25.63, 22.05, 19.54, 13.91. HRMS
(HESI+): calculated for [M]+ C20H36N+ (m/z): 290.28423, found: 290.2835.
1-Hexadecyl-2-methylpyridinium bromide (6c). Compound 6c was obtained as white crystals (80%); m.p.
126.8 ◦C–128.8 ◦C. 1H-NMR (500 MHz, DMSO)
δ
(ppm): 9.02 (d, J = 6.1 Hz, 1H), 8.51–8.44 (m, 1H),
8.05 (d, J = 7.9 Hz, 1H), 8.00–7.94 (m, 1H), 4.57–4.50 (t, J = 6.8 Hz, 2H), 2.84 (s, 3H), 1.88–1.78 (m, 2H),
1.58–1.06 (m, 26 H), 0.85 (t, J = 6.8 Hz, 3H); 13C-NMR (126 MHz, DMSO)
(ppm): 155.06, 145.32, 145.01,
δ
129.93, 125.54, 57.23, 31.26, 29.36, 29.02, 28.98, 28.91, 28.81, 28.68, 28.44, 25.65, 22.07, 19.53, 13.93. HRMS
(HESI+) calculated for [M]+ C22H40N+ (m/z): 318.31553; found: 318.3153.
3.1.2. Determination of Lipophilicity Expressed as Clog k or Clog p
Capacity factors k of the final products were measured with the aforementioned HPLC-MS system
using the isocratic method described by Benkova et al. (2019) [26], with the ratio of ACN:H2O (both
with 0.1% formic acid v/v) changed to 73:27. The studied compounds were dissolved in methanol.
Uracil (Sigma Aldrich, Steinheim, Germany) was used as a void volume marker (t0). Retention times
were determined from mass spectrometry total ion current scans. Capacity factors k were calculated
using the equation:
t1 − t0
k =
(1)
t0
where t1 = retention time of the analyte and t0 = void time. Uracil was used as a void volume marker.
Lipophilicity expressed as Clog p was calculated using MarvinSketch (Marvin 17.21.0, ChemAxon,
Budapest, Hungary, https://www.chemaxon.com), and the values were correlated to log k values.
Lipophilicity was expressed as a dependence of log k to the calculated Clog p, and the results in the
studied group were characterized by classical statistical criteria: coefficient of determination R2 (0.97+).

Molecules 2020, 25, 2254
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3.1.3. Conductivity Measurements
Conductivity measurements of aqueous solutions of the prepared surfactants were carried out in
triplicate on a Tristar Orion conductivity meter with conductivity cell 013005MD (Thermo Scientific,
Waltham, MA, USA). The apparatus was controlled by the Navigator 21 software, using continuous
data collection. The experiment was set up according to previous experience: the solutions were
temperature-controlled at 45
±
0.1 ◦C [
9]. Aqueous stock solutions of surfactants were prepared at
concentrations of 0.1500 mol/L for C12 homologs, 0.0200 mol/L for C14 homologs and 0.0150 mol/L for
C16. For the determination of CMC, the aqueous stock solutions of surfactants were further diluted.
The linear pump Lineomat (VEB MLW Labortechnik Ilmenau, Ilmenau, Germany) was used with a
flow rate of 0.43 mL/min. An AREX stirrer (VELP Scientifica Srl, Milano, Italy) was used for continuous
agitation, and conductivity was recorded every 3 s. The equation for the linear regression and the
coefficient of determination was calculated for both linear parts, and the intersection of both axes was
determined as the value of CMC (Figure S1).
3.2. The biological Evaluation of Picolinium Salts
3.2.1. Evaluation of Antibacterial Activity
The antibacterial activities of the prepared compounds were investigated on nine bacterial
strains, i.e., three Gram-positive (Staphylococcus aureus C1947, methicillin-resistant S. aureus C1926,
vancomycin-resistant enterococci S2484), and six Gram-negative (Escherichia coli A1235, Klebsiella
pneumoniae C1950, extended-spectrum
β-lactamase-producing K. pneumoniae C1934, Acinetobacter
baumannii J3474, Stenotrophomonas maltophilia J3552 and Yersinia bercovieri CNCTC 6230). The latter was
ordered from the Czech National Collection of Type Cultures (CNCTC), while the others were mostly
resistant clinical isolates of patients from the University Hospital in Hradec Kralove (Czech Republic).
The antibacterial properties and the minimum inhibitory (MIC) and minimum bactericidal
(MBC) concentrations of the compounds tested were determined by the broth microdilution testing
method modified according to standard M07-A11 [58] and the protocol published previously [26].
Mueller-Hinton broth (M-H broth, HiMedia, Mumbai, India) adjusted to pH 7.4 ( 0.2) was used
±
as the test medium. All compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich,
St. Louis, MO, USA) so that the final concentration of DMSO did not exceed 1% in the test medium
(also applied to growth control). The wells of the microtitration plates contained 200
broth with two-fold serial dilutions of the compounds (500; 250; 125; 62.5; 31.25; 15.63; 7.81; 3.91; 1.95;
0.98; 0.49 mol/L), and were inoculated with 10 L of bacterial suspension. A bacterial inoculum in
µL of the M-H
µ
µ
sterile water (B. Braun Medical s.r.o., Melsungen, Germany) was adjusted densitometrically to match
0.5 McFarland turbidity scale. The MIC values, defined as complete inhibition of bacterial growth,
were read visually and verified by microscopy after 24 h and 48 h incubation at 37 ◦C
±
1 ◦C. The MBCs
99.9% decrease in
µL of fresh M-H broth.
were established for all prepared compounds as the concentrations that provided
the number of colonies after subculturing a 10 L aliquot from each well in 200
≥
µ
The total incubation time was 48 h. For comparison, standard reference compounds N-benzalkonium
bromide (4B, 5B, 6B) were included in the corresponding concentrations.
3.2.2. Evaluation of Antifungal Activity
The antifungal activity was evaluated on seven fungal strains, i.e., four yeasts (Candida
parapsilosis sensu stricto EXF-8411, Rhodotorula mucilaginosa EXF-8417 and Exophiala dermatitidis EXF-8470,
Aureobasidium melanogenum EXF-8432), and three filamentous fungi (Bisifusarium dimerum EXF-8427,
Penicillium chrysogenum EXF-1818, and Aspergillus versicolor EXF-8692). The strains were obtained from
the Ex Culture collection (in the frame of the Infrastructural Centre Mycosmo) at the Department of
Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia. All isolates were maintained
on malt extract agar (MEA) prior to being tested.

Molecules 2020, 25, 2254
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The antifungal activities were evaluated using the agar-well diffusion test according to the adjusted
protocol published by Zovko et al. [59]. The fungal strain in the exponential (log) phase of growth was
inoculated into 10 mL of sterile liquid malt extract broth (MEB), and 100
was poured onto solid MEA and spread on the plate surface. After the suspension dried, the circles
of agar ( = 5.5 mm) were cut out from the inoculated culture plates. Then, 50 L of the prepared
µL of the prepared suspension
Φ
µ
compounds diluted in DMSO was added into the wells in the agar plates. Each concentration (1, 0.5,
0.3, 0.1, 0.03, 0.01 mmol/L) of the compounds was tested three times using three separate agar plates.
Plates were incubated at room temperature, and after 72 h (yeasts) or 7 days (filamentous fungi) of
incubation, the minimum inhibitory concentrations (MICs), defined as the lowest concentrations which
inhibit the visible growth of the microorganisms being tested, were determined. The diameters of the
inhibition zones were measured with a ruler. The shortest distance (0.5 mm) from the outer margin of
the well to the initial point of microbial growth was estimated as the inhibitory zone. The inhibitory
activity of DMSO was also checked and found to be zero.
3.2.3. Evaluation of Virucidal Activity
The virucidal activity of synthesized agents was evaluated using a strain of the varicella zoster
virus (VZV) from the collection of VZV isolates at the Department of Epidemiology, Faculty of Military
Health Sciences in Hradec Kralove, University of Defence in Brno, Czech Republic.
A quantitative suspension test was used to determine the virucidal activity of the tested compounds
against VZV. Briefly, one part of the virus suspension was mixed with one part of fetal bovine serum
diluted with phosphate-buffered saline (1:5.5) and eight parts of the prepared compounds or reference
drugs 4B
,
5B
,
6B. The mixtures were kept at room temperature for 5 min (exposure time). After the
L of each
exposure period, the mixtures were serially diluted ten-fold with ice-cold DMEM. Then, 100
µ
dilution were seeded into a 96-well microtiter plate with NHLF cells (ATCC, Manassas, VA, USA) and
incubated at 37 ◦C in a 5% CO2 atmosphere until a cytopathic effect was detected, i.e., approximately
10–12 days. Six wells per sample dilution were inoculated. Virus-untreated controls with identical
protein concentrations were also tested.
Virus titer was determined using the method of Spearman and Kaerber and expressed as log10
TCID50/mL including the standard [60]. According to the European Standard EN 14476 for virucidal
activity of disinfection, a product should demonstrate at least a log10 reduction of 4 in virus titer,
corresponding to 99.99% inactivation [61]. To differentiate between virus-induced cytopathogenic
changes and the toxic effect caused by the tested compounds, the cell monolayer was monitored
until the end of the virucidal activity testing period for morphological changes after exposure to the
disinfectant solution.
3.2.4. Cell Viability Assessment
Standard MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma
Aldrich, St. Louis, MO, USA) was used according to the manufacturer’s protocol on CHO-K1 cells
(Chinese hamster ovary, ECACC, Salisbury, UK) in order to compare the cytotoxic effect of the studied
compounds [26]. 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 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 0.5 mg/mL of MTT, and the cells
were allowed to produce formazan for approximately 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 a 650 nm reference wavelength on Synergy HT (BioTek, Winooski, VT,
USA). IC50 was then calculated from the control-subtracted triplicates using nonlinear regression (four

Molecules 2020, 25, 2254
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parameters) in the GraphPad Prism 5.03 and 7.03 software (GraphPad Software Inc., San Diego, CA,
USA). Final IC50 and SEM values were obtained as a mean of three independent measurements.
4. Conclusions
Picolinium salts with an aliphatic carbon chain (C12–C16) were prepared by one-step synthesis.
The products were characterized by common analytical methods (NMR and HRMS) and their biological
activities, lipophilicity and critical micellar concentration (CMC) were evaluated. The prepared
picolinium salts showed good antimicrobial properties as well as broad-spectrum activity against a
selected panel of microorganisms including bacteria, fungi and enveloped viruses below their CMC
values. Their activities were compared with the antimicrobial effect of commonly used benzalkonium
analogues with the same alkyl chain length. Picolinium salts with C14 and C16 side chains achieved
similar or better results for antimicrobial efficacy than the reference benzalkoniums. Their antibacterial
activity was comparable to that of selected standards on both G+ and G
the antibacterial effect was weaker. The best activity was shown against Staphylococcus aureus
(G+), while for G strains, the greatest efficiency was observed against Acinetobacter baumannii and
−
bacteria, although against
G
−
−
Yersinia bercovieri. Furthermore, they showed promising antifungal efficiency. Concurrently, the
picolinium salts exhibited satisfactory low cytotoxicity against mammalian cells, i.e., comparable to
that of benzalkonium compounds, which are considered as safe.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/9/2254/
s1. Figure S1: Dependency of CMC-specific conductivity on concentration; Figure S2: The broad-spectrum
antimicrobial activity; Table S1: Antibacterial activity; Table S2: Antifungal activity; Table S3: Antiviral activity;
Table S4.: Cytotoxicity and calculated lipophilicity
Author Contributions: Sa.S. and D.M. were responsible for the preparation of picolinium salts and characterization
of their structure and purity; Sa.S. and J.M. assessed the critical micellar concentration of prepared QAS; L.P. and
R.D. performed HPLC analysis including calculation of lipophilicity; R.S. assessed the antiviral activity; M.B.
and O.S. were responsible for the evaluation of QAS antibacterial and antifungal properties and cytotoxicity
assessment; K.S. and N.G.-C. took part in antifungal evaluation; K.K. and O.S. participated in the QAS design,
management of the study and final correction of the manuscript and correspondence. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was supported by the Czech Health Research Council (no. NV19-09-00198).
Acknowledgments: The authors are grateful to Ian McColl MD for assistance with the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AB
Acinetobacter baumannii
ACN
ASVE
AUME
BIDI
acetonitrile
Aspergillus versicolor
Aureobasidium melanogenum
Bisifusarium dimerum
BL
Klebsiella pneumoniae
CAPA
CMC
EC
Candida parapsilosis sensu stricto
critical micelle concentration
Escherichia coli
En
Vancomycin-resistant enterococci
extended spectrum β-lactamase producing Klebsiella pneumoniae
Exophiala dermatitidis
high-performance liquid chromatography coupled with mass spectrometry
high resolution mass spectrometry
minimum inhibitory concentration
minimum bactericidal concentration;
Methicillin-resistant Staphylococcus aureus
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
ESBL
EXDE
HPLC-MS
HRMS
MIC
MBC
MRSA
MTT

Molecules 2020, 25, 2254
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NMR
PECH
QAS
RHMU
SA
SEM
SE
SM
nuclear magnetic resonance
Penicillium chrysogenum
quaternary ammonium salts
Rhodotorula mucilaginosa
Staphylococcus aureus
standard deviation
Staphylococcus epidermis
Stenotrophomonas maltophilia
Varicella zoster virus
Yersinia bercovieri
VZV
YB
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