Synthesis, Structure and Antimicrobial Properties of Novel Benzalkonium Chloride Analogues with Pyridine Rings

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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 Pozna´n,Poland; [email protected] 2 Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Wólcza´nska171/173, 90-924 Łód´z,Poland; [email protected] (A.K.); [email protected] (A.O.) * Correspondence: [email protected]; 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 Figure1, 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, they arewere widely presented used for as the disinfectants first time as antimicrobial and antiseptics, agents as by well Domagk as preservatives in 1935 [13]. Nowadays, for ophthalmic, they are nasal widely used as disinfectants and antiseptics, as well as preservatives for ophthalmic, nasal and and inhalationalMolecules 2017, 22 drugs., 130 They are also active substances in medicines for throat and mouth2 infections. 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 most bacteria, fungi and algae is based on adsorption of of thewere alkylammonium presented for the cation first time on as the antimicrobial bacterial cell agents surface, by Domagk diffusion in 1935 through [13]. Nowadays, the cell wall they and are then widelythe alkylammonium used as disinfectants cation on and the antiseptics, bacterial cell as suwellrface, as diffusionpreservatives through for ophthalmic,the cell wall nasal and thenand binding and disruption of cytoplasmatic membrane. Damage to the membrane results in a release of inhalationalbinding and drugs. disruption They of are cytoplasmatic also active substances membrane. in Damagemedicines to for the throat membrane and mouth results infections. in a release The of potassium ions and other cytoplasmatic constituents, ﬁnally leading to the death of the cell [14–17]. mechanismpotassium ions of their and biocidalother cytoplasmatic action toward constituents, most bacteria, finally fungi leading and algaeto the is death based of on the adsorption cell [14–17]. 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.Examples Examples of of antimicrobial antimicrobial quaternaryquaternary ammonium ammonium halides: halides: alkylpyridinium alkylpyridinium (1); (1); alkyltrimethylammoniumalkyltrimethylammonium (2); (2 benzalkonium); benzalkonium ( 3(3);); dialkyldimethylammoniumdialkyldimethylammonium (4) (and4) and diesteralkonium 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,certain microorganism in recent years strains there to the have biocidal been activi frequentty of benzalkonium reports of cases chlorides of increasing [18–21]. The resistance use of of alkyltrimethylammonium (2); benzalkonium (3); dialkyldimethylammonium (4) and diesteralkonium (5). certaininappropriate microorganism concentrations strains toof benzalkonium the biocidalactivity chlorides of and benzalkonium incomplete removal chlorides of residues [18–21 of]. these The use of inappropriatemicrobiocidesHowever, concentrations afterin recent finishing years the there of disinfection benzalkonium have been pr frequentocess chlorides exposes reports andmicroorganisms of incompletecases of increasing to removal prolonged resistance of contact residues of of thesecertainwith microbiocides sublethal microorganism doses after of strains finishingBACs. toThis the the contributes biocidal disinfection activi to thety processdevelopmentof benzalkonium exposes of defensive chlorides microorganisms mechanisms [18–21]. The to and use prolonged the of contactinappropriateemergence with sublethal of concentrationsstrains doses of microorganisms of of BACs. benzalkonium This exhibiting contributes chlorides resistanceto and the incomplete developmentto benzalkonium removal of chloridesdefensiveof residues as mechanismsof well these as cross-resistance to certain other antibiotics [22,23]. This fact creates a serious epidemiological threat, and themicrobiocides emergence after of strainsfinishing of the microorganisms disinfection process exhibiting exposes resistance microorganisms to benzalkonium to prolonged chloridescontact as especially in medical facilities, manifested by an increase in morbidity and mortality among patients wellwith as cross-resistance sublethal doses toof BACs. certain This other contributes antibiotics to the [22 development,23]. This fact of createsdefensive a seriousmechanisms epidemiological and the [24]. One of the way to overcome this serious negative side effect is a periodically application of new threat,emergence especially of strains in medical of microorganisms facilities, manifested exhibiting by resistance an increase to benzalkonium in morbidity chlorides and mortality as well as among cross-resistancemicrobiocides with to certain modified other structures. antibiotics [22,23]. This fact creates a serious epidemiological threat, patients [24]. One of the way to overcome this serious negative side effect is a periodically application especiallyTo meet in medical this challenge facilities, we manifested have proposed by an aincrease modification in morbidity of benzalkonium and mortality chlorides among based patients on of new microbiocides with modified structures. [24].the introduction One of the way of nitrogen to overcome atoms this into serious the aromatic negative ring. side For effect that is reason a periodically a series of application novel quaternary of new microbiocidesToammonium meet this compounds challengewith modified weP13– havestructures.P18 with proposed variable a modificationalkyl chain length of benzalkonium and bearing pyridine chlorides rings based instead on the introductionof phenylTo meet of rings nitrogen this were challenge synthesized. atoms we into have In the proposedthe aromatic literatu are mo ring. theredification Forare thatknown of benzalkonium reason nonionic a series surfactants chlorides of novel containing based quaternary on ammoniumthe4-pyridyl introduction compounds moieties, of nitrogen forP13 example–P18 atomswith amines into variable the [25–27], aromatic alkyl amides ring. chain For[28], length that ethers reason and and bearinga ketonesseries of pyridine [29,30],novel quaternary as rings well insteadas of phenylammonium4-alkylpyridines rings compounds were [31]. synthesized. According P13–P18 to Inwith our the variable knowledge literature alkyl this there chain is the are length first known reportand bearing nonionic however pyridine surfactantsabout ringsthe synthesis instead containing of a series of benzalkonium chloride analogues bearing 4-pyridyl rings. 4-pyridylof phenyl moieties, rings were for examplesynthesized. amines In the [ 25literatu–27],re amides there are [28 known], ethers nonionic and ketones surfactants [29 ,containing30], as well as 4-pyridyl moieties, for example amines [25–27], amides [28], ethers and ketones [29,30], as well as 4-alkylpyridines2. Results and [31 Discussion]. According to our knowledge this is the first report however about the synthesis of a series4-alkylpyridines of benzalkonium [31]. According chloride to our analogues knowledge bearing this is4-pyridyl the first report rings. however about the synthesis of a seriesThe novel of benzalkonium compounds P13chloride–P18 analogueswere obtained bearing according 4-pyridyl to Scheme rings. 1 by Menshutkin reaction of 2. Results4-chloromethylpyridine and Discussion (6) with the corresponding N,N-dimethylalkylamines 7–12 containing 8, 10, 2.12, Results 14, 16, 18and carbon Discussion atoms in alkyl chain, respectively. The use of the polar aprotic solvent acetonitrile The novel compounds P13–P18 were obtained according to Scheme1 by Menshutkin reaction of as a reactionThe novel medium compounds allowed P13 us–P18 to wereobtain obtained compounds according P13–P18 to Scheme in good 1 yield by Menshutkin at room temperature. reaction of 4-chloromethylpyridine4-chloromethylpyridine (6 )(6 with) with the the corresponding corresponding NN,N,N-dimethylalkylamines-dimethylalkylamines 7–127 –containing12 containing 8, 10, 8, 10, 7, P13 n=5 12, 14,12, 16, 14, 18 16, carbon 18 carbon atoms atoms in in alkyl alkyl chain, chain, respectively.respectively. The The use use of of the the polar polar aprotic aprotic solvent solvent acetonitrile acetonitrile N N e 8, P14 n=7 as a reaction medium allowed us to obtain compoundsCH3CN P13–P18 in good yield at room temperature. as a reaction medium+ allowedN us to obtain compounds P13–P18 in good Clyield at room 9,temperature. P15 n=9 Cl r.t. a N n c g i 10, P16 n=11 b d f n h 11,7, P13 P17 n=5n=13 N 6 7-12N e P13-P18 12,8, P14 P18 n=7n=15 CH3CN + N Cl 9, P15 n=9 Cl r.t. a N Scheme 1.n Synthesis of compounds P13c –P18. g i 10, P16 n=11 b d f n h 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 1H-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 (Scheme1). Protons H-d and H-e of then methylene and methyl groups directly attached to the quaternary nitrogen atom appear in 1H-NMR spectra as singlets in the 5.30–5.24 ppm and 1 3.54–3.52 ppm regions, respectively. In all H-NMR spectra of the analyzed surfactants P13–P18 H2O proton signals are observed in the range 2.98–2.65 ppm. According to literature data the protons of H2O 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 13C-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 H2O 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 1H-NMR, 13C-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 Figure2. The heats of formation (HOF) and geometric parameters of compounds P13–P18 are presented in Table1. 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 (Table1).

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 someBDDAC extent1 to theBDDAB parameters2 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 ... − BDDAC 1 BDDAB 2 BDDAB 3 N(2) X 3.137 3.137P13 3.137P14 P15 3.137 P16 3.137P17 3.137P18 3.148 3.535 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 −25.638 1.502 −36.512 1.502 −47.387 1.502 −58.264 1.502−69.141 −44.786 1.502 −37.285 1.510 - 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) 1.525 1.525 1.525 1.525 1.525 1.525 1.525 1.526 1.538 N(2)–C(1) 1.508 1.508 1.508 1.508 1.508 1.508 1.508 1.515 1.517 C(3)–C(13)N(2)–C(4) 1.494 1.494 1.502 1.502 1.494 1.502 1.494 1.502 1.4941.502 1.4941.502 1.502 1.494 1.510 1.492 1.531 1.487 C(13)–C(14)N(2)–C(3) 1.397 1.397 1.536 1.536 1.397 1.536 1.397 1.536 1.3971.536 1.3971.536 1.537 1.397 1.539 1.397 1.546 1.402 C(14)–C(15)N(2)–C(5) 1.394 1.394 1.529 1.529 1.394 1.529 1.394 1.529 1.3941.529 1.3941.529 1.528 1.390 1.534 1.389 1.510 1.393 C(15)–N(16)C(5)–C(6) 1.353 1.353 1.525 1.525 1.353 1.525 1.353 1.525 1.3531.525 1.3531.525 1.525 - 1.526 - 1.538 - N(16)–C(17)C(3)–C(13) 1.352 1.352 1.494 1.494 1.352 1.494 1.352 1.494 1.3521.494 1.3521.494 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 1.3971.397 1.397 1.391 1.397 1.392 1.402 1.356 C(13)–C(18)C(14)–C(15) 1.396 1.396 1.394 1.394 1.396 1.394 1.396 1.394 1.3961.394 1.3961.394 1.390 1.397 1.389 1.398 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) 108.556 108.558 1.397 108.5581.397 1.397 108.560 1.397 108.558 1.397 108.5581.397 108.556 1.391 1.392 109.668 1.356 109.560 C(1)–N(2)–C(3)C(13)–C(18) 107.159 107.159 1.396 107.1571.396 1.396 107.158 1.396 107.158 1.396 107.1591.396 107.223 1.397 1.398 108.079 1.363 108.440 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)C(1)–N(2)–C(5) 110.391 110.393 110.034 110.035 110.395 110.034 110.392 110.034 110.393 110.035 110.393110.035 109.989110.446 108.868 109.140 110.100 112.530 C(4)–N(2)–C(5)C(3)–N(2)–C(4) 110.593 110.593 110.028 110.024 110.594 110.024 110.593 110.024 110.594 110.024 110.593110.023 109.982110.566 112.142 108.890 108.160 107.990 N(2)–C(3)–C(13)C(3)–N(2)–C(5) 114.561 114.562 110.391 110.393 114.564 110.395 114.562 110.392 114.562 110.393 114.561110.393 110.446114.413 109.140 113.849 112.530 116.070 N(2)–C(5)–C(6)C(4)–N(2)–C(5) 112.170 112.171 110.593 110.593 112.171 110.594 112.171 110.593 112.171 110.594 112.170110.593 110.566112.243 108.890 112.948 107.990 116.240 Dihedral [◦]N(2)–C(3)–C(13) 114.561 114.562 114.564 114.562 114.562 114.561 114.413 113.849 116.070 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)N(2)–C(3)–C(13)–C(14)−93.233 −93.232 90.143 −90.14593.240 90.140−93.233 90.145− 93.24190.139 −93.23390.146 − 90.13893.370 89.914−93.929 89.500 −89.540 C(4)–N(2)–C(3)–C(13)N(2)–C(3)–C(13)–C(18) 58.616 58.621−93.233 − 58.63093.232 −93.240 58.620 −93.233 58.632 −93.241 58.626−93.233 − 60.17693.370 −93.929 44.503 −89.540 60.950 C(5)–N(2)–C(3)–C(13)C(4)–N(2)–C(3)–C(13)−63.720 −63.715 58.616 −58.62163.707 58.630−63.715 58.620− 63.70458.632 −63.70958.626 − 60.17662.133 44.503−76.241 60.950 −58.260 C(1)–N(2)–C(3)–C(13)C(5)–N(2)–C(3)–C(13) 176.460 176.463−63.720 − 176.47163.715 −63.707 176.465 −63.715 176.474 −63.704 176.469−63.709 − 178.03062.133 −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 2 3 benzyldimethyldodecylammonium1 benzyldimethyldodecylammonium chloride; chloride; 2benzyldimethyldodecylammonium benzyldimethyldodecylammonium bromide; 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 This allowsphase. The one heat to of extrapolateformation (HOF) some of the conclusions synthesized compounds from the decreases calculated linearly structure with an increasing to the condensed number of methylene groups in the hydrocarbon chain (Figure 3a). The higher the number of methylene phase. Theunits, heat the lower of formation the heat of formation. (HOF) of There the is synthesizedalso a good correlation compounds between decreasesthe melting points linearly of with an increasingN number,N-dimethyl- ofN methylene-(4-methylpyridyl)- groupsN-alkylammonium in the hydrocarbon chlorides chain and the (Figure number3 a).of methylene The higher groups the number of methylenein their units, alkyl thechains lower (Figure the 3a). heat The ofhigher formation. melting points There reflect is alsothe better a good packing correlation in the crystal between the melting pointswhat is of aN result,N-dimethyl- of the strongN-(4-methylpyridyl)- hydrophobic interactionsN-alkylammonium between straight hydrocarbon chlorides chains. and the An number of observed increasing stability of N,N-dimethyl-N-(4-methylpyridyl)-N-alkylammonium chlorides methylenewith groups the lengthening in their alkyl of the chains alkyl chain (Figure is in a3 a).good The accordance higher with melting the increasing points reﬂect melting the points better of packing in the crystalthese what compounds, is a result which ofreflect the a strong sigmoidal hydrophobic type of correlation interactions (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 (Figure3b). Molecules 2017, 22, 130 5 of 12 Molecules 2017, 22, 130 5 of 12

-10 HOF 105 -20 melting points 100 95 -30 90 -40 C)

85 o

-50 t ( 80

HOF (kcal/mol) -60 75 -70 70 -80 65 8 1012141618 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 ofof theP13– hydrocarbonP18. chain of compounds P13–P18; Plot (b) The correlation of melting points vs. HOF of P13–P18. 2.3. Aggregation Behavior 2.3. AggregationThe aggregation Behavior behavior of the synthesized surfactants was evaluated by a conductometric study 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 ◦ performedsurfactants. in aqueous A common solutions method at 25 ofC. estimation Critical micellizationof CMC values concentrations from conductivity (CMCs) vs. surfactant for compounds P14–P18concentrationwere obtained plots fromis by thefinding plots the of intersection specific conductivity point of two (leastκ) against squares the method concentrations fitted lines of the surfactants.assigned A for common pre- and methodpost-micellar of estimationregions. Unfortunat of CMCely, this values method from is not conductivity suitable for all vs. types surfactant of concentrationsurfactants, plots as is in by some finding cases thean inflection intersection point point is very of difficult two least to find squares because method of the low fitted curvature lines assigned for pre-of and the post-micellar plots. In such cases, regions. the measurements Unfortunately, are thissubject method to large is errors. not suitable In the case for of all the types synthesized of surfactants, compounds, their CMCs were difficult to estimate by the conventional method. For that reason as in somecritical cases micellization an inflection concentrations point isfor very compounds difficult P14 to–P18 find were because obtained of by the the low analysis curvature of the of the plots. Inplots such of differential cases, the conductivity measurements (dκ/dc) are vs. subjectsurfactant to concentration. large errors. This In is the more case precise ofthe approach synthesized compounds,that has their been CMCs successfully were difficultapplied in to determining estimate by CMC the values conventional [36]. method. For that reason critical micellizationThe concentrations plots of dκ/dc vs. for concentration compounds possessP14– reverseP18 were sigmoid obtained shapes byand the thus analysis can be fitted of the to a plots of differentialBoltzmann-type conductivity sigmoid (dκ/dc) expressed vs. surfactant by the following concentration. equation: This is more precise approach that has been successfully applied in determining CMC values − [36]. = + (1) The plots of dκ/dc vs. concentration possess reverse1+ sigmoid shapes and thus can be fitted to a Boltzmann-typeParameters sigmoid A1 and expressed A2 represent by the the following pre-micell equation:ar 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.A P141 −–P17A2 display CMC values typical of cationic y = A2 + − (1) surfactants that decrease significantly with increasing alkylx chainx0 length, because of the increase of the 1 + e dx hydrophobic character of the surfactants. In the case of P18 the increase of alkyl chain length causes a Parametersless significant A1and change A2 in represent CMC values the probably pre-micellar because and of coiling post-micellar of the long slopes, alkyl chains respectively. in water [37]. The width of transition, dx, possesses a central point, x , corresponding to the CMC value (Figure4). CMC values Table 2. CMC values and Klevens equation0 parameters of P14–P18, benzalkonium chlorides (BAC) for compoundsand trimethylammoniumP14–P18 are given chlorides in (TMAC). Table2 . 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 alkyl chain length N 1 10 12 14 16 18 A B R 2 causes a less significantP14–P18 change87.12 in20.17 CMC 4.87 values 1.14 probably 0.310 because 1.9978 of coiling 0.3073 of the 0.9996 long alkyl chains in 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 and1 N = Klevensalkyl chain equation length; 2 Ref. parameters [37]; 3 Ref. of[38];P14 4 Ref.–P18 [39];, benzalkonium 5 Ref. [40]. chlorides (BAC) and trimethylammonium chlorides (TMAC).

CMC [mM] Klevens Equation Parameters

N 1 10 12 14 16 18 A B R 2 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 = x = 0.00114 mol/dm3 0.00150 0 1.0 Molecules 2017, 22, 130 6 of 12

0.00125 0.9 CMC = x = 0.00114 mol/dm3 0.00150 0 1.0 0.00100 0.8 0.00125 0.9 /mol] 2 0.00075 0.7 x [S/dm] 0.00100 0 0.8 κ

0.00050 0.6 /dc [S*dm κ /mol] d 2 0.00075 0.7 x 0.00025 [S/dm] 0 0.5 κ

0.00050 0.6 /dc [S*dm κ d 0.00000 0.4 0.00025 0.0004 0.0008 0.0012 0.0016 0.00200.5 3 C (mol/dm ) FigureFigure 4. Specific 4. Specific conductivity conductivity0.00000 (• (•)) and and differentialdifferential conductivity conductivity Boltzman-type Boltzman-type fitting fitting0.4 (―) (vs.— )concentration vs. concentration of P17 at◦ 25 °C 0.0004 0.0008 0.0012 0.0016 0.0020 of P17 at 25 C. 3 C (mol/dm ) TheFigure novel 4. Specific compounds conductivity possess (•) and CMCs differenti muchal conductivity higher than Boltzman-type benzalkonium fitting chlorides(―) vs. concentration bearing alkyl chainsThe novelof of P17 the at compounds same25 °C length. possessThe data CMCsobtained much from higherFigure 5 than and benzalkoniumTable 2 indicate chloridesthat the compounds bearing alkyl chainspresented of the samein this length.paper exhibit The data mice obtainedllization behavior from Figure more similar5 and Tableto that2 ofindicate trimethylalkylammonium that the compounds presentedchloridesThe in this novelrather paper compoundsthan exhibit to BACs. micellizationpossess The KlevensCMCs behaviormuch equation higher more (2) than is similar often benzalkonium used to that to ofcharacterize trimethylalkylammoniumchlorides bearingthe relation alkyl chloridesbetweenchains rather of the the CMC thansame of tolength. a BACs.surfactant The Thedata and Klevensobtained its structure from equation [4Figure1]. It (2) presents5 and is often Table a linear used 2 indicate relationship to characterize that the between compounds the the relation betweenlogpresented CMC the CMCand in this alkyl of paper a chain surfactant exhibit length mice andof thellization its surfactant: structure behavior [41 more]. It similar presents to that a linear of trimethylalkylammonium 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 A is a constant that reflects the free energy change related to transfer of hydrophilic part of the log CMC and alkyl chain length of the surfactant:= − surfactant from the aqueous phase to thelogCMC micelle, whereasA BN parameter B determines the free energy (2) logCMC = A − BN (2) change associated with the transfer of a methylene (−CH2−) unit of the hydrophobic chain from aqueous wheresolutionwhere A is aA into constantis a theconstant micelle. that that reﬂects N reflects is the the number the free freeenergy ofenergy carbon changech atomsange related relatedin alkyl to tochain transfer transfer of surfactant.of hydrophilic of hydrophilic part partof the of the surfactantsurfactant from from the the aqueous aqueous phase phase to to the the micelle, micelle, whereas parameter parameter B determines B determines the free the freeenergy energy change associated with2.1 the transfer of a methylene (−CH −) unit of the hydrophobic chain from change associated with the transfer of a methylene (−CH2−) unit2 of the hydrophobicBAC chain from aqueous aqueoussolution solution into the into micelle. the micelle. N is the N number is the numberof carbon of atoms carbon in alkyl atoms chain in alkylof surfactant. chain of surfactant. 1.7 TMAC 1.3 P14-P18 2.1 BAC 0.9 1.7 TMAC 0.5 1.3 P14-P18 0.1

Log CMC 0.9 -0.3 0.5 -0.7 0.1 Log CMC -1.1 -0.3 8 101214161820 -0.7 N -1.1 Figure 5. Graphical presentation of the Klevens equation for P14–P18, TMAC and BAC (N = alkyl 8 101214161820 chain length). N The value of B obtained from linear fitting of the Klevens equation (Table 2) allowed us to determine FigureFigure 5. Graphical 5. Graphical presentation presentation of of the the Klevens Klevens equation for for P14P14–P18–P18, TMAC, TMAC and and BAC BAC (N = (N alkyl = alkyl the change of the Gibbs free energy of methylene group transfer, ΔG(−CH2−), according to the equation: chainchain length). length). ΔG() = −2.3RTB (3) The value of B obtained from linear fitting of the Klevens equation (Table 2) allowed us to determine Thethe change value ofof Bthe obtained Gibbs free from energy linear of fittingmethylene of the group Klevens transfer, equation ΔG(−CH2 (Table−), according2) allowed to the us equation: 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 (Table3). 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 (Figure6). 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 (Table3). 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 (Figure7). 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 (Table4). 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 observedMolecules for 2017 C16,, 22, 130 i.e., at a slightly higher chain length than for benzalkonium chlorides.8 of Because 12 of thevalues low solubilityare observed of for longer C16, alkyli.e., at derivativesa slightly higher we werechain unablelength than to check for benzalkonium their MIC and chlorides. compare it withvaluesBecause BACs. are of In theobserved general low solubility for benzalkonium C16, of i.e. longer, at a alkyl slightly chlorides derivatives higher show chainweslightly were length unable lowerthan to forcheck MIC benzalkonium their values MIC in and comparisonchlorides. compare to the pyridylBecauseit with BAC analogues,of thes. Inlow general solubility however benzalkonium of longer for Pseudomonas alkyl chlorides derivatives show aeruginosa we slightly werethe lowerunable C16 MIC to pyridyl check values their analoguein MICcomparison and showed compare to the better it with BACs. In general benzalkonium chlorides show slightly lower MIC values in comparison to the antimicrobialpyridyl analogues, activity (Table however4). for Pseudomonas aeruginosa the C16 pyridyl analogue showed better pyridylantimicrobial analogues, activity however (Table 4). for Pseudomonas aeruginosa the C16 pyridyl analogue showed better 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] N 1 N 1 MIC [mM] N 1 P.P. aeruginosa aeruginosa A. A.niger niger C. albicans C. albicans P. aeruginosa A. niger C. albicans 8 8 55 5 52.5 2.5 10 108 1.251.255 0.6255 0.625 0.3122.5 0.312 12 1012 0.3120.3121.25 0.07810.625 0.0781 0.3120.039 0.039 14 0.0781 0.0195 0.0097 1214 0.07810.312 0.07810.0195 0.00970.039 16 0.156 0.156 0.0195 1416 0.07810.156 0.01950.156 0.00970.0195 1 N = alkyl chain length. 16 0.1561 N = alkyl chain0.156 length. 0.0195 1 N = alkyl chain length. 13 12.4970 12.4999 1.5625 1312 1.5 A. niger 12.4970 12.4999 1.5625 1211 1.5 A.C. nigeralbicans 11 C. albicans 10 1 P. chrysogenum 109 1 P. chrysogenum

MIC [mM] MIC 0.7182 98 0.5[mM] MIC 0.7182 87 0.5 0.3906 7 6 0.39060.1953 0.1953 0.0976 MIC [mM] MIC 65 0 0.1953 0.1953 0.0976 MIC [mM] MIC 54 0 12 14 16 43 3.1250 12 14 16 32 3.1250 21 10 0 8 10 12 14 16 8 10 12N 14 16 N FigureFigure 6. 6Antifungal. Antifungal activity activity of P13––P17P17 (N(N = =alkyl alkyl chain chain length). length). Figure 6. Antifungal activity of P13–P17 (N = alkyl chain length). 0.8 0.7812 S. aureus 6.2485 0.8 6 0.7 0.7812 6.2485 S.B. aureus subtilis 6 0.7 0.6 B.E. subtiliscoli 0.6 5 0.5 E.P. coliaeruginosa 0.5 5 0.4 0.3906 P. aeruginosa

4 0.40.3 0.3906 MIC [mM] MIC 4 0.3 MIC [mM] MIC 0.2 0.1953 0.1953 0.1953 3 0.20.1 0.1953 0.0977 0.0976 0.0488 0.0488

MIC [mM] MIC 0.0976 3 0.10 0.0977 0.0244 0.0488 0.0488 MIC [mM] MIC 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 12N 14 16 N Figure 7. Antibacterial activity of P13–P17 (N = alkyl chain length). FigureFigure 7. 7.Antibacterial Antibacterial activityactivity of of P13P13––P17P17 (N(N = alkyl = alkyl chain chain length). 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 (Pozna´n, Poland) except for N,N-dimethyloctadecylamine that was purchased from TCI (Łód´z, 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 (Gda´nsk, 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 CH3CN (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%; ◦ −1 1 m.p. 67–70 C; IR (KBr) νmax 3057, 2929, 2850, 1596, 1473 cm ; 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), 13 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); 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%; ◦ −1 1 m.p. 72–75 C; IR (KBr) νmax 3023, 2921, 2850, 1601, 1470 cm ; 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, 13 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); 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%; ◦ -1 1 m.p. 83–86 C; IR (KBr) νmax 3062, 2920, 2850, 1602, 1470 cm ; 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), 13 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); 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%; ◦ -1 1 m.p. 91–93 C; IR (KBr) νmax 3062, 2915, 2850, 1602, 1470 cm ; 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), 13 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); 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%; ◦ -1 1 m.p. 96–98 C; IR (KBr) νmax 3053, 2921, 2850, 1601, 1470 cm ; 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), 13 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); 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. ◦ -1 1 100–102 C; IR (KBr) νmax 3054, 2915, 2851, 1600, 1472 cm ; 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, 13 H-g), 1.17–1.35 (30 H, m, H-h), 0.87 (3 H, t, J = 6.4, 7.3 Hz, H-i); 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, Gda´nsk, 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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