Synthesis and antimicrobial properties of binaphthyl derived quaternary ammonium bromides

European Journal of Pharmaceutical Sciences 65 (2014) 29–37

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier .com/ locate/e jps

Synthesis, surface and antimicrobial properties of some quaternaryammonium homochiral camphor sulfonamides

http://dx.doi.org/10.1016/j.ejps.2014.08.0130928-0987/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Comenius University in Bratislava, Faculty of Phar-macy, Department of Chemical Theory of Drugs, Odbojárov 10, 83232 Bratislava,Slovakia. Tel.: +421 250117323.

E-mail address: [email protected] (R. Mikláš).

R. Mikláš a,⇑, N. Miklášová a, M. Bukovsky b, B. Horváth c, J. Kubincová a, F. Devínsky a

a Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Slovakiab Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Slovakiac NMR Laboratory, Faculty of Pharmacy, Comenius University in Bratislava, Slovakia

a r t i c l e i n f o

Article history:Received 26 May 2014Received in revised form 15 August 2014Accepted 30 August 2014Available online 10 September 2014

Keywords:Quaternary ammonium saltsAntimicrobial activityHomochiral camphorsulfonamidesCritical micelle concentration

a b s t r a c t

A group of homochiral quaternary ammonium sulfonamides bearing hydrophobic camphor derived moi-eties were synthesized and characterized. The described synthetic procedure is quick and efficient. Thenovel quaternary ammonium bromides were tested as antimicrobial and antifungal agents. They exhib-ited strong antimicrobial and also antifungal activity, especially N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicy-clo[2.2.1]heptan-1-yl)methylsulfonamido] ethyl}-N,N-dimethyltetradecan-1-aminium bromide 1c. Thesurface properties of prepared compounds were evaluated by surface tension measurements and criticalmicelle concentration (CMC) with surface tension at CMC (cCMC) was calculated.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Since the quaternary ammonium salts (QUATs) were preparedfor the first time by Menschutkin (1890) in the reaction of tertiaryamine with alkyl halides they had been studied during the 20thcentury thoroughly. Practical applications of quaternary ammo-nium salts had been found in textile finishes (excellent fabric soft-eners), antielectrostatic agents and wood preservatives (Gilbert andMoore, 2005; Kim and Sun, 2002), catalysts (Shirakawa et al., 2012;Brak and Jacobsen 2013; Bilé et al., 2012) and since 1998 also asionic liquids (Welton, 1999). Since it was found that cationic lipids,known as cytofectins, are efficient for delivering functional genes(Brigham et al., 1989) the use of cationic amphiphiles for mediatingDNA transfection has increased (Sajomsang et al., 2013; de lasCuevas et al., 2012; Cortesi et al., 2012). The strong bactericidalactivity of QUATs with long alkyl chains have been known from1915 (Jacobs and Heidelberger, 1915) and studied further on abroad range of microorganisms such as bacteria (both G+ and G�)and fungi (Merianos 1991; Lukác et al., 2010; Struga et al., 2008;Pernak and Chvala, 2003; Mikláš et al., 2012; Tischer et al., 2012),certain viruses (Wong et al., 2002) and even anticancer agents(Yip et al., 2006; Simeone et al., 2012). Development of resistance

in microorganisms toward disinfectants or antibiotics (Heinzel,1988; Morente et al., 2013) brings the necessity to supply recentlyapplied antimicrobial agents by new, potent and safe ones and thussearch for new and effective molecules goes on (Feder-Kubis andTomczuk, 2013; Hoque et al., 2012; Semenov et al., 2011;Colomer et al., 2011; Chanawanno et al., 2010). The QUATs, withone long alkyl chain at least, belong to amphiphilic compounds.These salts possess properties such as reduction of surface tensionan also the attraction for negatively charged bacteria surface. Hav-ing the ability to intercalate into phospholipid membranes theymay affect the processes in biological systems inducing cell autoly-sis leading to the leakage of intercellular materials into the environ-ment and cell death (Kopecká-Leitmanová et al., 1989; Devinskyet al., 1987; Mlynarcík et al., 1981). The mode of action of cationicsurfactants on bacteria’s membrane strongly depends on ability toform micelles and intercalate into the bacterial membrane becausethe disruption of membrane and subsequent solubilization of itsparts plays a crucial role in the cell death process. The antimicrobialeffect of QUATs is bilinearly related to their micellization propertieswhich are linearly related to the length of the alkyl chains. The anti-microbial activity for Gram-positive bacteria is optimal when themaximum of the carbon chain length is C12–C14 while for Gram-negative bacteria activity is increased with the chain length ofC14–C16 (Thebault et al., 2009; Pernak and Skrzypczak, 1996;Devinsky et al., 1990, 1991). The highest antimicrobial activityshows QUATs with critical micelle concentration range 1 � 10�2 to1 � 10�4 mol L�1 (Devínsky et al., 1985). Molecules with n-alkyl

30 R. Mikláš et al. / European Journal of Pharmaceutical Sciences 65 (2014) 29–37

chain length below C4 and above C18 are antimicrobially ineffec-tive. In the series of structurally related QUATs the antimicrobialactivity increases with the growing chain length until the maxi-mum. From this point with continuous growing chain length theantimicrobial activity starts to decrease. This phenomenon, calledcut-off effect, is typical for many of the biologically active com-pounds and it can be caused by many reasons (limited aqueous sol-ubility, kinetic effects, interaction with lipid bilayers or proteins)(Balgavy and Devínsky, 1996). The antimicrobial activity in fact isaffected not only by alkyl chain length but also by other hydropho-bic groups in the molecule. The study of this effect on antimicrobialproperties could help in development of new active QUATs. In addi-tion, introduction of new structural motives such as heteroatoms oraromatics (Semenov et al., 2011; Pernak et al., 2001) may result inpotential antimicrobial agents with higher biological activities andthey could even conquer the growing resistance phenomenon. Thewell-known antibacterial effect of essential oils containing bicycli-cal camphor or borneol (Ruiz-Navajas et al., 2012; Miguel et al.,2011) brought us to the idea to design and synthesize QUATs bear-ing hydrophobic camphor derived sulfonamides, hoping that incor-poration of two important antimicrobially active structures in onecompound will improve their bioactivity. The introduction of anester group into the molecule contributes to better biodegradabilityof such compounds and in fact compounds 1e–1g, and 2e–2gbelong to the so called ‘‘soft’’ quaternary ammonium type antimi-crobials (Bodor et al., 1980; Bodor and Kaminski 1980). Moreoverwe strongly believe that looking for new highly effective antimicro-bials is very important especially nowadays when new highly resis-tant bacterial stems are emerging (Buffet-Bataillon et al., 2012).

In this study we have prepared as illustrated in Fig. 1 series of 14new optically active quaternary ammonium salts with camphorsulfonamides and tested their antimicrobial activity against Gram-negative Escherichia coli, Gram-positive human pathogenic bacteriaStaphylococcus aureus and human fungal pathogen Candida albicans.Their surface properties were studied by surface tension measure-ments. The critical micelle concentration was calculated from theplot of logarithm of the surfactant‘s concentration vs. surface tension.

2. Experimental procedure

2.1. Materials and methods

All compounds used ((1S)-(+)-camphor-10-sulfonic acid (CSA),thionylchloride, diethyl ether, acetone, dichloromethane (DCM),

Fig. 1. Synthesis of QUATs derived from

ethyl acetate, DMSO, bromoalkanes) are commercially available.DCM was pre-dried over CaCl2 and then distilled from CaH2 undernitrogen atmosphere. Diethyl ether was distilled from sodiumprior to use. Bromoacetic acid esters were synthesized by modifi-cation of already published procedure (Nguyen et al., 2004). A cor-responding alcohol and bromoacetic acid were heated in thepresence of toluene-4-sulfonic acid in toluene with an azeotropicremoval of water.

1H and 13C NMR spectra were measured on a Varian Gemini 300spectrometer at 300 MHz and 75 MHz respectively. Chemical shiftshave been reported in ppm relative to an internal reference (TMS).IR spectra (in KBr pellets) were recorded on FTIR Impact 400DNicolet instrument. Polarimetric measurements were obtainedusing a Jasco P-1010 polarimeter at 589 nm. Elemental analyseswere carried out on a Carlo Erba 1108A instrument. HR-MS analy-ses were made in high-resolution system LTQ Orbitrap XL (ThermoFisher Scientific, San Jose, CA, USA). Samples were ionized by elek-trospray ION MAX-ESI in positive mode, respectively. Conditionsfor positive ionization were: spray voltage 4 kV, sheath gas 5 arbi-trary units, capillary temperature 280 �C, capillary voltage 30 V,lens voltage 90 V. Data were scanned during 1 min. in fullscanmode at the mass range m/z 80–1000 with a resolution of 30,000(at m/z 400). The system was calibrated before analysis to an exter-nal standard and samples were dosed by infusion system at a flowrate of 2,5 ll/min.

All melting points reported were uncorrected and measured onKofler hot stage. The surface tension measurements were per-formed on Krüss processor tensiometer K100 (Wilhelmy platemethod). Temperature was kept constant at the desired level usingthermostatted (Thermo Haake SC100) water bath. Double-distilledwater was used for the preparation of all samples. Measurementsof equilibrium surface tension were taken repeatedly until thechange in surface tension was less than 0.08 mN m�1. The valuesof surface tension decrease with increasing concentration and thebreak point provides the CMC value and surface tension at CMC(cCMC).

2.2. Microbiology

The antimicrobial activity was tested against Gram-negativebacteria E. coli CNCTC 377/79, Gram-positive bacteria S. aureusATCC 6538 and fungi C. albicans CCM 8186. Solutions of com-pounds studied were prepared in DMSO (5%). A suspension of thestandard microorganism, prepared from 24 h cultures of bacteriain blood agar and from 24 h cultures in the Sabouraud agar for

(1S)-(+)-camphor-10-sulfonic acid.

R. Mikláš et al. / European Journal of Pharmaceutical Sciences 65 (2014) 29–37 31

fungi had a concentration of 5 � 107 cfu mL�1 of bacteria and5 � 105 cfu mL�1 of Candida. Concentration of microorganismswere determined spectrophotometrically at 540 nm and adjustedto absorbance A = 0.35. The microorganism suspension was addedto solutions containing the tested compound and to double con-centrated peptone broth medium (8%) for bacteria or Sabouraudmedium (12%) for Candida. The stock solution of tested compoundswas serially diluted by half. The cultures were done in 96-wellmicroliter plates. The microorganisms were incubated for 24 h at37 �C and then from each well 5 microliters of suspension werecultured on blood agar (bacteria) or on Sabouraud agar (fungi).After 24 h at 37 �C the lowest concentration of QUATs which pre-vented colony formation was determined as minimal inhibitoryconcentration (MIC). (Lukác et al., 2010) As a standard benzalkoni-um bromide (BAB, Ajatin�) was used.

2.3. Synthesis

Enantiopure camphor sulfonylchloride 5 was prepared accord-ing to the known procedure (Gayet et al., 2004).

2.3.1. N-(2-dimethylaminoethyl)-1-[(1S)-7,7-dimethyl-2-oxobicyclo[2.1.1]heptan-1-yl]methanesulfonamide (3)

To the solution of N,N-dimethylethane-1,2-diamine (2.18 ml,0.04 mol) in anhydrous DCM (25 ml) was added dropwise a solu-tion of 5 (10 g, 0.04 mol) in anhydrous DCM (30 ml) at 0 �C during30 min. After the addition was complete the reaction mixture washeated subsequently to reflux for 2 h. and then mixed over night atambient temperature. The solvent was removed on rotary evapora-tor and 50 ml of dry diethyl ether was added to the residue. Theresulting suspension was mixed 30 min and filtered. A white pow-der of 3.HCl thus obtained was washed twice with 25 ml of drydiethyl ether and crystallized from acetone/methanol (10:1, v/v).A 3.HCl was prepared in 90% yield. Free base 3 was eliberated bydropwise addition of 10% aqueous NaOH into the suspension of3.HCl in ethyl acetate at 0 �C until the pH was adjusted to 12. Aque-ous layer was separated and washed 3 times with 30 ml of ethylacetate. The combined organic layers were dried over anhydrousNa2SO4 and evaporated to yield yellowish oil (97%). The productwas used in the next reaction without further purification. 1HNMR (CDCl3, 300 MHz) d 0.90 (s, 3H, CH3); 1.06 (s, 3H, CH3); 1.44(m, 1H, CH); 1.81–1.91 (m, 1H); 1.96–2.08 (m, 2H); 2.12 (t, 1H,J = 4.68 Hz); 2.23 (s, 6H, N(CH3)2); 2.28–2.56 (m, 4H); 2.93 (d, 1H,CH2–SO2, J = 14.94 Hz); 3.17–3.31 (m, 2H); 3.55 (d, 1H, CH2–SO2,J = 14.94 Hz); 5.45 (s, 1H, NH).

2.3.2. N-(4-methylpiperazin-1-yl)-1-[(1S)-7,7-dimethyl-2-oxobicyclo[2.1.1]heptan-1-yl]methane sulfonamide (4)

Compound 4 was prepared by the same procedure described for3 by the reaction of N-methylpiperazine (4.96 g, 0.05 mol) withsulfonylchloride 5 (12.4 g, 0.05 mol) in DCM. Sulfonamide 4 wasisolated as a white solid in 65% yield. M.p. 151–153 �C. 1H NMR(CDCl3, 300 MHz) d 0.88 (s, 3H, CH3); 1.13 (s, 3H, CH3); 1.38–1.47(m, 1H); 1.60–1.70 (m, 1H); 1.94 (d, 1H, J = 18.4 Hz); 1.99–2.12(m, 2H); 2.32 (s, 3H, CH3–N); 2.33–2.57 (m, 6H); 2.74 (d, 1H,J = 14.36 Hz); 3.32–3.36 (m, 5H).

2.3.3. General procedure for the synthesis of quaternary salts 1and 2

10 mmol of sulfonamide 3 or 4 were mixed with 1.3 equivalentof alkylating bromoderivative in CH3CN (25 ml). Reaction mixturewas stirred at ambient temperature for 2 h, then refluxed for16 h (series a–d) or 8 h (series e–g) respectively and cooled. Thereaction mixture was placed in the freezer overnight and whitecrystals were filtered off, washed twice with 25 ml of anhydrous

diethyl ether and the crude product was recrystallized repeatedlyfrom anhydrous acetone-methanol mixture.

2.3.4. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-N,N-dimethyldecan-1-aminium bromide (1a)

Yield 76%, colorless crystals, mp 115–117 �C. [a]D20 = +28.6

(1.0 g l�1, CHCl3) 1H NMR (CDCl3, 300 MHz) d 0.88 (m, 6H). 1.06(s, 3H); 1.25–1.51 (m, 16H); 1.72–2.12 (m, 5H); 2.25–2.41 (m,2H); 3.03 (d, 1H, J = 14.83 Hz); 3.41 (s, 6H); 3.47–3.59 (m, 3H);3.80–3.95 (m, 4H); 7.29 (t, 1H, J = 6.04 Hz). 13C NMR (CDCl3,75 MHz) d 215.76; 65.86; 63.38; 58.54; 52.14; 49.05; 48.66;42.84; 42.79; 38.25; 31.96; 29.56; 29.52; 29.37; 29.33; 27.13;26.33; 25.30; 22.91; 22.78; 19.98; 19.76; 14.24. IR m/cm�1 3433,3062, 3006, 2922, 2852, 1747, 1469, 1333, 1151, 1054, 785. Ele-mental Anal. Calcd. for C24H47BrN2O3S: C 55.05, H 9.05, N 5.35,S 6.12. Found: C 54.14, H 8.72, N 5.08, S 6.65. HRMS for C24H47N2O3S(M + H)+, calcd 443.3302, found 443.3302.

2.3.5. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-N,N-dimethyldodecan-1-aminium bromide (1b)

Yield 68%, colorless crystals, mp 98–101 �C. [a]D20 = +15.6

(1.0 g l�1, CHCl3) 1H NMR (CDCl3, 300 MHz) d 0.87 (m, 6H). 1.05(s, 3H); 1.24–1.50 (m, 18H); 1.75–2.11 (m, 7H); 2.30–2.39 (m,2H); 3.00 (d, 1H, J = 14.65 Hz); 3.40 (s, 6H); 3.46–3.56 (m, 3H);3.69–3.94 (m, 4H); 7.24 (t, 1H, J = 5.86 Hz). 13C NMR (CDCl3,75 MHz) d 215.71; 65.85; 63.38; 58.55; 52.14; 49.10; 48.66;42.85; 42.79; 38.24; 31.93; 29.56; 29.52; 29.44; 29.37; 29.33;27.13; 26.34; 25.30; 22.91; 22.80; 19.98; 19.77; 14.24. IR m/cm�1

3433, 3062, 3006, 2922, 2852, 1747, 1469, 1333, 1151, 1054,785. Elemental Anal. Calcd. for C26H51BrN2O3S: C 56.61, H 9.32, N5.08, S 5.81. Found: C 56.06, H 9.03, N 4.80, S 6.41. HRMS forC26H51N2O3S (M + H)+, calcd 471.3615, found 471.3606.

2.3.6. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfonamido]ethyl}-N,N-dimethyltetradecan-1-aminium bromide (1c)

Yield 65%, colorless crystals, mp 136–138 �C. [a]D20 = +18.2

(1.0 g l�1, CHCl3) 1H NMR (CDCl3, 300 MHz) d 0.88 (m, 6H). 1.05(s, 3H); 1.25–1.51 (m, 22H); 1.77–2.10 (m, 7H); 2.32–2.39 (m,2H); 3.00 (d, 1H, J = 14.65 Hz); 3.40 (s, 6H); 3.47–3.57 (m, 3H);3.82–3.91 (m, 4H); 7.22 (t, 1H, J = 5.27 Hz).). 13C NMR (CDCl3,75 MHz) d. 215.65; 65.71; 63.24; 58.41; 52.10; 48.95; 48.93;48.54; 48.52; 48.50; 42.71; 42.67; 31.90; 29.71; 29.69; 29.66;29.60; 29.54; 29.43; 29.36; 29.23; 27.00; 26.22; 25.17; 22.78;22.69; 19.87; 19.65; 14.14. IR m/cm�1 3433, 3062, 3006, 2922,2852, 1747, 1469, 1333, 1151, 1054, 785. Elemental Anal. Calcd.for C28H55BrN2O3S: C 58.01, H 9.56, N 4.83, S 5.53. Found: C56.80, H 9.28, N 4.40, S 6.11. HRMS for C28H55N2O3S (M + H)+, calcd499.3928, found 499.3919.

2.3.7. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-N,N-dimethylhexadecan-1-aminium bromide (1d)

Yield 74%, colorless crystals, mp 105–106 �C. [a]D20 = +33.0

(1.0 g l�1, CHCl3) 1H NMR (CDCl3, 300 MHz) d 0.88 (m, 6H). 1.06(s, 3H); 1.19–1.51 (m, 28H); 1.73–2.12 (m, 5H); 2.25–2.41 (m,2H); 3.02 (d, 1H, J = 14.65 Hz); 3.41 (s, 6H); 3.47–3.57 (m, 3H);3.84 (d, 4H, J = 25.7 Hz); 7.20 (t, 1H, J = 5.75 Hz). 13C NMR (CDCl3,75 MHz) d 215.57; 65.73; 63.24; 58.41; 52.01; 48.94; 48.93;48.54; 48.53; 48.50; 42.71; 42.67; 31.90; 29.70; 29.69; 29.68;29.66; 29.60; 29.54; 29.43; 29.36; 29.23; 27.00; 26.22; 25.17;22.78; 22.69; 19.87; 19.65; 14.14. IR m/cm�1 3433, 3061, 3007,2922, 2851, 1746, 1469, 1334, 1151, 1053, 785. Elemental Anal.Calcd. for C30H59BrN2O3S: C 59.29, H 9.78, N 4.61, S 5.28. Found:

32 R. Mikláš et al. / European Journal of Pharmaceutical Sciences 65 (2014) 29–37

C 58.22, H 9.52, N 4.45, S 5.95. HRMS for C30H59N2O3S (M + H)+,calcd 527.4241, found 527.4232.

2.3.8. Quaternary salt 2-(decyloxy)-N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfonamido]ethyl}-N,N-dimethyl-2-oxoethanaminium bromide (1e)

Yield 52%, colorless crystals, mp 134–138 �C. [a]D20 = +10.3

(1.0 g l�1, CHCl3) 1H NMR (CDCl3, 300 MHz) d 0.88 (m, 6H). 1.07(s, 3H); 1.28 (s, 16H); 1.45–1,53 (m, 1H); 1.68 (t, 2H, J = 5.88 Hz);1.89–2.12 (m, 4H); 2.28–2.39 (m, 2H); 2.96 (d, 1H, J = 14.64 Hz);3.48 (d, 1H, J = 14.64 Hz); 3.68 (s, 6H); 3.85 (d, 2H, J = 4.2 Hz);4.07–4.24 (m, 4H); 4.87 (s, 2H); 7.09 (t, 1H, J = 5.8 Hz). 13C NMR(CDCl3, 75 MHz) d 215.75; 164.68; 67.11; 64.78; 61.65; 58.38;53.22; 48.90; 48.74; 42.75; 42.68; 38.48; 31.96; 29.75; 29.69;29.65; 29.52, 29.41; 29.24; 28.25; 27.11; 25.66; 25.25; 22.71;19.87; 19.62; 14.26. IR m/cm�1 3434, 3065, 2942, 2851, 1747,1463, 1334, 1150, 1053, 785. Elemental Anal. Calcd. forC26H49BrN2O5S: C 53.69, H 8.49, N 4.82, S 5.51. Found: C 51.86, H8.15, N 4.24, S 5.80. HRMS for C26H49N2O5S (M + H)+, calcd501.3357, found 501.3349.

2.3.9. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-2-(dodecyloxy)-N,N-dimethyl-2-oxoethanaminium bromide (1f)

Yield 48%, colorless crystals, mp 119–120 �C. [a]D20 = +20.7

(1.0 g l�1, CHCl3) 1H NMR (CDCl3, 300 MHz) d 0.88 (m, 6H). 1.05(s, 3H); 1.26 (s, 20H); 1.44–1,51 (m, 1H); 1.68 (t, 2H, J = 5.86 Hz);1.90–2.12 (m, 4H); 2.28–2.40 (m, 2H); 2.96 (d, 1H, J = 14.65 Hz);3.48 (d, 1H, J = 14.65 Hz); 3.67 (s, 6H); 3.84 (d, 2H, J = 4.1 Hz);4.07–4.22 (m, 4H); 4.87 (s, 2H); 7.05 (t, 1H, J = 5.8 Hz). 13C NMR(CDCl3, 75 MHz) d 215.75; 164.50; 67.06; 64.74; 61.61; 58.45;53.25; 48.82; 48.64; 42.73; 42.69; 38.41; 31.93; 29.73; 29.68;29.66; 29.63; 29.51, 29.38; 29.23; 28.24; 27.04; 25.65; 25.24;22.71; 19.87; 19.62; 14.16. IR m/cm�1 3436, 3065, 2956, 2924,2853, 1749, 1457, 1336, 1236, 1205, 1150, 785. Elemental Anal.Calcd. for C28H53BrN2O5S: C 55.16, H 8.76, N 4.59, S 5.26. Found:C 54.59, H 8.61, N 4.17, S 6.01. HRMS for C28H53N2O5S (M + H)+,calcd 529.3670, found 529.3659.

2.3.10. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-N,N-dimethyl-2-oxo-2-(tetradecyloxy)ethanaminium bromide (1g)

Yield 78%, colorless crystals, mp 133–134 �C. [a]D20 = +21.0

(1.0 g l�1, CHCl3). 1H NMR (CDCl3, 300 MHz) d 0.88 (m, 6H). 1.05(s, 3H); 1.26 (s, 24H); 1.43–1,51 (m, 1H); 1.66 (t, 2H, J = 6.87 Hz);1.87–2.11 (m, 4H); 2.28–2.39 (m, 2H); 2.97 (d, 1H, J = 14.83 Hz);3.49 (d, 1H, J = 14.83 Hz); 3.68 (s, 6H); 3.82 (d, 2H, J = 5.22 Hz);4.07–4.22 (m, 4H); 4.88 (s, 2H); 7.12 (t, 1H, J = 6.04 Hz). 13C NMR(CDCl3, 75 MHz) d 215.92; 164.65; 67.17; 64.85; 61.70; 58.56;53.36; 48.90; 48.75; 42.83; 42.79; 38.51; 32.04; 29.82; 29.79;29.75; 29.73; 29.63, 29.49; 29.38; 29.33; 28.35; 27.15; 27.13;25.78; 25.34; 22.82; 19.97; 19.72; 14.26.. IR m/cm�1 3434, 3062,2955, 2922, 2852, 1748, 1467, 1335, 1236, 1206, 1153, 786. Ele-mental Anal. Calcd. for C30H57BrN2O5S: C 56.50, H 9.01, N 4.39,S 5.03. Found: C 55.54, H 8.85, N 4.32, S 5.65. HRMS for C30H57N2O5S(M + H)+, calcd 557.3983, found 557.3973.

2.3.11. Quaternary salt 1-decyl-4-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methyl sulfonamido]-1-methylpiperazinium bromide (2a)

Yield 71%, colorless crystals, mp 225–226 �C. [a]D20 = +279.1

(0.99 g l�1, CHCl3). 1H NMR (CDCl3, 300 MHz) d 0.88 (t, 3H,J = 6.45 Hz); 0.92 (s, 3H);. 1.07 (s, 3H); 1.26–1.53 (m, 16H); 1.65–1.78 (m, 3H); 1.94–2.17 (m, 4H); 2.29–2.41 (m, 2H); 3.09 (d, 1H,J = 14.67 Hz); 3.54 (d, 1H, J = 15.25 Hz); 3.59 (s, 3H); 3.63–4.08(m, 8H). 13C NMR (CDCl3, 75 MHz) d 215.7; 65.1; 59.8; 59.7;

58.3; 48.9; 48.1; 47.3; 42.7; 42.6; 39.5; 31.8; 29.5; 29.4; 29.2;27.0; 26.2; 25.3; 22.6; 22.1; 19.9; 19.5; 14.1. IR m/cm�1 2923,2850, 1754, 1632, 1456, 1338, 1151, 1066, 1049, 780. ElementalAnal. Calcd. for C25H47BrN2O3S: C 56.06, H 8.84, N 5.23, S 5.99.Found: C 55.03, H 8.64, N 4.96, S 6.86. HRMS for C25H47N2O3S(M + H)+, calcd 455.3302, found 455.3294.

2.3.12. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-dodecyl-1-methylpiperazinium bromide (2b)

Yield 53%, colorless crystals, mp 228–229 �C. [a]D20 = +281.1

(0.99 g l�1, CHCl3). 1H NMR (CDCl3, 300 MHz) d 0.88 (t, 3H,J = 7.04 Hz); 0.92 (s, 3H);. 1.07 (s, 3H); 1.25–1.53 (m, 20H); 1.65–1.78 (m, 3H); 1.94–2.15 (m, 4H); 2.29–2.41 (m, 2H); 3.10 (d, 1H,J = 14.96 Hz); 3.55 (d, 1H, J = 15.25 Hz); 3.59 (s, 3H); 3.63–4.08(m, 8H). 13C NMR (CDCl3, 75 MHz) d 215.6; 65.1; 59.8; 59.7;58.3; 48.9; 48.0; 47.3; 42.7; 42.6; 39.5; 31.9; 29.6; 29.5; 29.4;29.3; 29.2; 27.0; 26.3; 25.3; 22.6; 22.1; 19.9; 19.5; 14.1. IRm/cm�1 2924, 2853, 1748, 1632, 1474, 1352, 1329, 1155, 1072,771. Elemental Anal. Calcd. for C27H51BrN2O3S: C 57.53, H 9.12, N4.97, S 5.69. Found: C 56.37, H 8.85, N 4.67, S 6.63. HRMS forC27H51N2O3S (M + H)+, calcd 483.3615, found 483.3609.

2.3.13. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-methyl-1-tetradecylpiperazinium bromide (2c)

Yield 48%, colorless crystals, mp 222–225 �C. [a]D20 = +252.9

(1.0 g l�1, CHCl3). 1H NMR (CDCl3, 300 MHz) d 0.88 (t, 3H,J = 7.04 Hz); 0.92 (s, 3H);. 1.07 (s, 3H); 1.25–1.53 (m, 24H); 1.66–1.87 (m, 3H); 1.94–2.13 (m, 4H); 2.30–2.39 (m, 2H); 3.10 (d, 1H,J = 15.25 Hz); 3.55 (d, 1H, J = 15.84 Hz); 3.59 (s, 3H); 3.64–4.08(m, 8H). 13C NMR (CDCl3, 75 MHz) d 215.6; 65.1; 59.8; 59.7;58.3; 48.9; 48.1; 47.3; 42.7; 42.6; 39.5; 31.9; 29.6; 29.5; 29.4;29.3; 29.2; 27.0; 26.2; 25.3; 22.6; 22.1; 19.9; 19.5; 14.1. IRm/cm�1 2923, 2853, 1747, 1632, 1468, 1350, 1331, 1155, 1068,1049, 779. Elemental Anal. Calcd. for C29H55BrN2O3S: C 58.86, H9.37, N 4.73, S 5.42. Found: C 57.65, H 9.02, N 4.57, S 5.89. HRMSfor C29H55N2O3S (M + H)+, calcd 511.3928, found 511.3919.

2.3.14. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-hexadecyl-1-methylpiperazinium bromide (2d)

Yield 53%, colorless crystals, mp 221–224 �C. [a]D20 = + 247.0

(1.0 g l�1, CHCl3). 1H NMR (CDCl3, 300 MHz) d 0.88 (t, 3H,J = 7.04 Hz); 0.92 (s, 3H);. 1.06 (s, 3H); 1.25–1.53 (m, 28H); 1.65–1.86 (m, 4H); 1.95–2.14 (m, 3H); 2.31–2.42 (m, 2H); 3.09 (d, 1H,J = 15.25 Hz); 3.54 (d, 1H, J = 15.25 Hz); 3.60 (s, 3H); 3.64–4.08(m, 8H). 13C NMR (CDCl3, 75 MHz) d 215.8; 65.1; 59.8; 59.7;58.3; 49.0; 48.2; 47.3; 42.7; 42.5; 39.5; 31.9; 29.7; 29.6; 29.6;29.5; 29.4; 29.3; 29.2; 27.0; 26.2; 25.2; 22.7; 22.1; 19.9; 19.5;14.1. IR m/cm�1 2923, 2852, 1748, 1632, 1468, 1351, 1330, 1157,1072, 773. Elemental Anal. Calcd. for C31H59BrN2O3S: C 60.07, H9.60, N 4.52, S 5.17. Found: C 59.15, H 9.45, N 4.39, S 5.59. HRMSfor C31H59N2O3S (M + H)+, calcd 539.4241, found 539.4230.

2.3.15. Quaternary salt 1-(decyloxycarbonylmethyl)-4-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1] heptan-1-yl)methylsulfonamido]-1-methylpiperazinium bromide (2e)

Yield 72%, colorless crystals, mp 186–189 �C. [a]D20 = +231.2

(1.0 g l�1, CHCl3). 1H NMR (CDCl3, 300 MHz) d 0.88 (t, 3H,J = 7.03 Hz); 0.92 (s, 3H);. 1.06 (s, 3H); 1.26 (s, 12H); 1.46–1.54(m, 1H); 1.63–1.74 (m, 3H); 1.97–2.14 (m, 4H); 2.29–2.40 (m,2H); 3.09 (d, 1H, J = 15.23 Hz); 3.55 (d, 1H, J = 15.23 Hz); 3.74–4.31 (m, 14H); 5.22 (s, 2H). 13C NMR (CDCl3, 75 MHz) d 215.8;164.5; 67.1; 60.9; 60.3; 58.4; 49.0; 48.2; 47.9; 42.7; 42.6; 39.4;39.3; 31.9; 29.5; 29.4; 29.3; 29.1; 28.2; 27.0; 25.6; 25.1; 22.7;

R. Mikláš et al. / European Journal of Pharmaceutical Sciences 65 (2014) 29–37 33

19.9; 19.5; 14.1. IR m/cm�1 2925, 2855, 1743, 1632, 1468, 1356,1336, 1218, 1158, 1072, 780, 710, 554. Elemental Anal. Calcd. forC27H49BrN2O5S: C 54.63, H 8.32, N 4.72, S 5.40. Found: C 53.47, H8.12, N 4.39, S 6.13. HRMS for C27H49N2O5S (M + H)+, calcd513.3357, found 513.3347.

2.3.16. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-(dodecyloxycarbonylmethyl)-1-methylpiperazinium bromide (2f)

Yield 69%, colorless crystals, mp 179–181 �C. [a]D20 = +228.7

(1.0 g l�1, CHCl3). 1H NMR (CDCl3, 300 MHz) d 0.89 (t, 3H,J = 7.02 Hz); 0.93 (s, 3H);. 1.07 (s, 3H); 1.27 (s, 16H); 1.49–1.56(m, 1H); 1.69 (t, 3H, J = 8.20 Hz); 1.86 (s, 1H); 2.00–2.14 (m, 3H);2.31–2.41 (m, 2H); 3.09 (d, 1H, J = 15.23 Hz); 3.55 (d, 1H,J = 15.23 Hz); 3.81–4.28 (m, 14H); 5.25 (s, 2H). 13C NMR (CDCl3,75 MHz) d 216.0; 164.5; 67.1; 60.7; 60.4; 60.3; 58.4; 49.0; 48.2;47.9; 42.7; 42.5; 39.4; 39.3; 31.9; 29.6; 29.5; 29.3; 29.1; 28.2;27.0; 25.6; 25.1; 22.7; 19.9; 19.5; 14.1. IR m/cm�1 2924, 2853,1750, 1632, 1469, 1344, 1329, 1220, 1156, 1069, 781, 714, 563.Elemental Anal. Calcd. for C29H53BrN2O5S: C 56.02, H 8.59, N4.51, S 5.16. Found: C 54.98, H 8.42, N 4.27, S 5.73. HRMS forC29H53N2O5S (M + H)+, calcd 541.3670, found 541.3660.

2.3.17. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-methyl-1-(tetradecyloxycarbonylmethyl)piperazinium bromide (2g)

Yield 79%, colorless crystals, mp 174–176 �C. [a]D20 = +212.2

(1.0 g l�1, CHCl3). 1H NMR (CDCl3, 300 MHz) d 0.88 (t, 3H,J = 7.03 Hz); 0.91 (s, 3H); 1.06 (s, 3H); 1.26 (s, 20H); 1.48–1.54(m, 1H); 1.66–1.71 (m, 3H); 1.98–2.12 (m, 4H); 2.30–2.40 (m,2H); 3.09 (d, 1H, J = 14.64 Hz); 3.55 (d, 1H, J = 15.23 Hz); 3.79–4.28 (m, 14H); 5.22 (s, 2H). 13C NMR (CDCl3, 75 MHz) d 215.9;164.5; 67.1; 60.9; 60.3; 58.4; 49.0; 48.2; 47.9; 42.7; 42.6; 39.4;39.3; 31.9; 29.6; 29.5; 29.3; 29.1; 28.2; 27.0; 25.6; 25.1; 22.7;19.9; 19.5; 14.1. IR m/cm�1 2924, 2853, 1749, 1632, 1469, 1343,1329, 1221, 1156, 1070, 781, 714, 563. Elemental Anal. Calcd. forC31H57BrN2O5S: C 57.30, H 8.84, N 4.31, S 4.93. Found: C 56.28, H8.60, N 3.87, S 5.52. HRMS for C31H57N2O5S (M + H)+, calcd569.3983, found 569.3971.

2.4. Stability studies

Studied compounds (5 mg) were dissolved in PBS solution(0.70 ml, D2O) and placed into NMR tubes. The progress of hydro-lysis was monitored by 1H NMR. Spectra were taken at the givenperiod of time for the samples kept at 37 �C. PBS buffer, pH 7.2was prepared by mixing of KH2PO4 (0.131 g) and Na2CO3

(0.067 g) in D2O (5 ml). PBS buffer, pH 2.5 was prepared by mixingof KH2PO4 (0.131 g) in D2O (5 ml) and adjusted to pH 2.5 by drop-wise addition of 10% HCl in H2O.

3. Results and discussion

Enantiopure quaternary ammonium salts 1 and 2 were synthe-sized as illustrated in Fig. 1. starting from (1S)-camphor-10-sul-fonic acid. Although sulfonylchloride 5 is commercially availableas a starting material, (1S)-camphor-10-sulfonic acid proved tobe less expensive and can be easily converted to the sulfonylchlo-ride 5 by the procedure already published (Gayet et al., 2004). Thus(1S)-camphor-10-sulfonic acid was reacted with thionylchlorideproviding compound 5 in 86% yield after crystallization frompetroleum ether. The preparation of camphor sulfonamides 3 and4 was carried out by drop wise addition of equimolar amount of5 in anhydrous DCM into the solution of N,N-dimethylethan-1,2-diamine and N-methylpiperazine in DCM respectively. The

presence of tertiary amino group in the structure allowed us toomit the use of another base for binding hydrochloric acid formedduring the reaction. The hydrochlorides 3.HCl and 4.HCl thusobtained were purified by crystallization. The free bases 3 and 4were eliberated by treatment with aqueous NaOH and quaternizedby appropriate bromoderivative in acetonitrile in order to give twoseries of QUATs 1 and 2. All quaternary ammonium sulfonamideswere obtained as colorless crystals after several crystallizationsfrom anhydrous acetone–methanol mixture in yields ranged from48 to 79%. They were identified and characterized thoroughly fromspectral and analytical data. The higher differences in elementalanalysis, mainly for carbon, are caused by hydrophilic nature ofprepared salts. Approximately 0.5–1 molecule of water per QUAT‘smolecule is present in the sample. HR-MS spectra clearly provedthe structure of synthesized QUATs.

Quaternary ammonium salts exhibit strong antimicrobial activ-ities and they are widely used as a disinfectants and antiseptics.The main target site of QUATs is the cytoplasmic membrane sur-rounding the cytoplasm of a cell and comprised of a phospholipidbilayer. QUATs are able to intercalate into phospholipid bilayerwhich is accompanied by membrane disorganization and struc-tural and functional changes in the cell wall inducing leakage ofintracellular components (Tischer et al., 2012; Hoque et al., 2012;Wessels and Ingmer, 2013; Gilbert and Moore, 2005). In additionQUATs were found to inhibit ATP synthesis by neutralizing the pro-ton motive force (PMF) (Denyer and Hugo, 1977). The PMF is initi-ated by a proton gradient across the cytoplasmic membrane and itis involved in many respiratory and photosynthetic processesincluding ATP synthesis. Quaternary ammonium salts are surfaceactive agents and therefore, they denature proteins anchored inthe cytoplasmic membrane or cause dissociation of an enzymefrom its prosthetic group. This effect was observed at concentra-tions much higher than lethal ones so the enzyme inhibition isnot the primary or main lesion caused by cationic surfactants(Merianos 1991). It has been shown that some bisammonium saltshave intracellular target and bind to DNA which leads to the inhi-bition of DNA replication (Zinchenko et al., 2004; Menzel et al.,2011). On the other hand, for most of the QUATs no specific targetsite has been recognized. However it is not excluded that there canexist some target specificities as shown by Menzel et al. (2011) andZhang et al. (2013) because the antimicrobial activity of QUATsfluctuates significantly against various types of microorganismsand explanation simply by the cationic charge and hydrophobictail cannot be used. The antimicrobial activities of the sulfonamideQUATs, were determined as a minimal inhibitory concentration(MIC, [lmol l�1]) against the Gram-positive human pathogenicbacteria S. Aureus, Gram-negative bacteria E. Coli and human fungalpathogen C. Albicans, the values for which are given in Table 1. TheMIC values were determined as lowest concentration of QUATsthat completely prevented visible colony formation. All the studieswere carried out in DMSO. In order to prove that the solvent doesnot influence bacterial and fungal growth a test with pure solventwas performed. This control test detected no inhibiting activity.Clinically used benzalkonium bromide (BAB, Ajatin�) was used asa standard.

Most of the prepared QUATs, except of 1a, 1e, 2a and 2g,showed higher antibacterial activity against Gram-positive bacte-ria S. aureus than standard BAB. The similar trend was observedalso in the activity against E. coli. The only compounds with loweractivity against E. coli compared to BAB were 1a, 2d and 2g. In thecase of C. albicans it was 1a, 1b, and 2a–2d. Activities of investi-gated compounds were slightly higher for Gram-positive bacteriathan for Gram-negative bacteria except of compound 1e whichexhibits the same antimicrobial activity for both Gram-positiveand Gram-negative bacteria. Surprisingly, the activity of soft salts1e–1g and 2e–2g against fungi was mainly similar or higher to that

Table 1MIC values (lmol l�1) for prepared QUATs.

Compound S. aureus ATCC6538

E. coli CNCTC 377/79

C. albicans CCM8186

1a 149.20 298.41 596.821b 8.85 17.69 35.401c 1.05 2.2 1.051d 2.01 128.54 4.011e 33.57 33.57 16.781f 8.02 16.04 8.021g 7.67 61.40 1.922a 45.60 182.20 182.22b 10.60 173.10 86.602c 5.20 41.20 164.902d 9.80 1260.40 315.102e 10.28 82.20 20.552f 5.63 78.40 2.452g 37.55 480.90 9.39BAB 26.00 260.00 26.00

34 R. Mikláš et al. / European Journal of Pharmaceutical Sciences 65 (2014) 29–37

against Gram-positive bacteria. The antimicrobial activity of theprepared compounds was also studied as a function of the numberof carbon atoms in the long alkyl chain. A bilinear dependence ofthe relative biological activity (log 1/MIC) on the length of thehydrophobic carbon chain (Figs. 2 and 3.), called cut-off effect,can be observed for almost all the synthesized compounds. Thecut-off effect is a general phenomenon and it has been observed

3,0

3,5

4,0

4,5

5,0

5,5

6,0

log

(1/M

IC)

10 11 12 13 14 15 16n

Fig. 2. Relation between the number of carbon atoms in alkyl chain (n) and antimicr

log

(1/M

IC)

4,2

4,4

4,6

4,8

5,0

5,2

5,4

5,6

5,8

log

(1/M

IC)

10 11 12 13 14n

Fig. 3. Relation between the number of carbon atoms in alkyl chain (n) and antimicrobaureus j E. colid C. albicans N.

in various biological and toxic activities in practically every amphi-phile homologous series tested. Several theories have been pro-posed to explain the cut-off effect. Ferguson (1939) was one ofthe first who has suggested that the cut-off effect in biologicalactivity could be caused by a decrease in the achievable compoundconcentration at the site of action due to its limited solubility.According to his model, the drug partition coefficient betweenthe aqueous solution and the site of action increases less rapidlywith the increasing chain length than the aqueous solubilitydecreases. Thus the compounds with longer hydrophobic chainshave limited partitioning which results in significantly lower con-centration at the site of action with no or small biological effect.Several authors (Ross et al., 1953; Birnie et al., 2000) have pro-posed that micellization is responsible for cut-off effect in biologi-cal activities of surfactant‘s type biocides. Also this theory is basedon decreased concentration of active compound at the site ofaction but due to the fact that the long chain surfactant moleculeshave higher predisposition for micellization than the tendency tomove toward the cell membrane (interface). From our previousstudies (Balgavy and Devínsky, 1996) we have proposed the freevolume hypothesis to explain the cut-off effect. In the bilayer,the amphiphile polar groups will interact with lipid polar groupsand their chains will orient parallel to the lipid hydrocarbonchains. At this location, the packing density of the bilayer hydro-phobic region must be influenced due to lateral expansion of thebilayer and formation of free volume close to the amphiphile chain

10 11 12 13 14 15 162,5

3,0

3,5

4,0

4,5

5,0

5,5

6,0

6,5

7,0

log

(1/M

IC)

n

obial activity of 1a–1d (left) and 2a–2d (right). S. aureus j E. colid C. albicans N.

10 11 12 13 143,23,43,63,84,04,24,44,64,85,05,25,45,6

n

ial activity of 1e–1g (left) and 2e–2g (right), CH2COO group is not counted in n. S.

R. Mikláš et al. / European Journal of Pharmaceutical Sciences 65 (2014) 29–37 35

ends. The bigger is the difference between the chain length ofphospholipids and amphiphile the bigger free volume is created.The free volume can be eliminated by rearrangement of hydrocar-bon chains (increased frequency of trans–gauche isomerization,bending and chain interdigitation) which results in the change ofthe bilayer thickness. The interaction of compounds with a shorthydrophobic chain creates a large free volume but the number ofmolecules built in the membrane is too small so the total free vol-ume created in the bilayer hydrophobic region will be small. As theamphiphile chain becomes long and comparable with lipid hydro-carbon chains the free volume will be small. This will result insmall changes in the membrane structure and low biologicalresponse. Amphiphilic molecules with chain length between theseextremes will thus induce maximal free volume and therefore theywill cause maximal membrane disruption accompanied by maxi-mal biological response. We should like to note that biological sys-tems are complicated and it is not excluded that the cut-off effectin these systems could be caused by a combination of differentmechanisms.

Table 2Cmc and ccmc values of quaternary ammonium salts 1a–1g at 25 �C.

Compound cmc (mold m�3) ccmc (mN m�1)

1a 4.39 ± 0.40 � 10�3 29.51b 2.62 ± 0.12 � 10�3 34.31c 1.31 ± 0.01 � 10�3 36.81d 0.81 ± 0.06 � 10�5 39.31e 1.91 ± 0.04 � 10�3 32.31f 0.72 ± 0.14 � 10�3 32.51g 2.71 ± 0.24 � 10�4 24.9

-6,5 -6,0 -5,5 -5,0 -4,5 -4,0 -3,5 -3,0 -2,5 -2,0 -1,5 -1,025

30

35

40

45

50

55

60

65

Surf

ace

tens

ion

(mN

m-1

)

1a1d

log c

-3,8 -3,6 -3,4 -3,2 -330

35

40

45

50

55

60

65

Surf

ace

tens

ion

(mN

m-1

)

Fig. 4. Surface tension versus the logarithm of the a

The highest inhibition activity of hard salts 1a–1d and 2a–2dagainst Gram-negative bacteria E. coli and Gram-positive bacteriaS. aureus was found for compounds with fourteen carbon atomsin alkyl chain. The behavior of fungi C. albicans was not so predict-able. In the series of compounds 1a–1d the highest fungicidalactivity was also observed for compounds with fourteen carbonatoms in alkyl chain. The piperazine derived salts 2a–2d exhibitedthe highest activity with their alkyl chain length being twelve car-bon atoms only. The series of soft quaternary salts 1e–1g and 2e–2g exhibit the best antimicrobial activity against Gram-positivebacteria S. aureus and Gram-negative bacteria E. coli with twelvecarbon atoms in the long alkyl chain (CH2COO group is notcounted). The best inhibition of C. albicans growth by soft saltswas also determined being twelve carbon atoms in alkyl chainfor piperazine derivatives 2e–2g but fourteen carbon atoms forsalts 1e-1g. The ‘‘soft’’ hydrolysable ester parts of the compounds1e–1g and 2e–2g, respectively, did not negatively influence theantimicrobial activity of such compounds.

The surface properties of prepared QUATs were investigated bysurface tension measurements. Quaternary ammonium salts 2a–2gexhibit low solubility in water. Thus only linear decreasing of sur-face tension was observed from the plots. To increase the solubilityand thus the desired concentration of compounds 2a–2g we havealso examined surface behavior at increased temperature (up to41 �C) but with no break in the plot. The determination of criticalmicelle concentration of compounds 1a–1g with more flexible eth-ylene spacer between camphor moiety and polar head was moresuccessful. Critical micelle concentrations of QUATs are shown inTable 2. The plots of surface tension against the logarithm ofsurfactant‘s concentration are presented in Fig. 4. It can be seenform Table 2 that values of the critical micelle concentration

-5,5 -5,0 -4,5 -4,0 -3,5 -3,0 -2,5 -2,0

25

30

35

40

45

50

551e1f1g

Surf

ace

tens

ion

(mN

m-1)

log c

,0 -2,8 -2,6 -2,4 -2,2 -2,0 -1,8

1b1c

log c

queous molar concentration of 1a–1g at 25 �C.

Fig. 5. Hydrolytic pathway of compounds 1e–1g.

Fig. 6. 1H NMR monitoring of 1e hydrolysis in PBS buffer, pH 7.2, at 37 �C.

36 R. Mikláš et al. / European Journal of Pharmaceutical Sciences 65 (2014) 29–37

decrease with number of carbon atoms in the chain beingincreased in both of the series 1a–1d and 1e–1g respectively.

To study the stability of prepared QUATs under physiologicaland acidic conditions 1H NMR technique was used. Quaternaryammonium salts 1 were subjected to hydrolysis (Fig. 5) in PBS/D2O buffer (pH 7.2) and PBS/HCl buffer (pH 2.5). The reactionswere performed at 37 �C in NMR tubes. As we expected, the salts1a–1d without an ester functional group, did not undergo hydroly-sis under studied conditions and NMR spectra showed no changesafter 480 h. The ‘‘soft’’ analogues 1e–1g with hydrolysable esterpart surprisingly showed good stability at pH 2.5 with no modifica-tion in NMR spectra after 480 h. As illustrated in Fig. 6, exposure ofammonium salt 1e to physiological pH 7.2 showed changes in NMRspectra almost instantly. The signals of protons H1, H2 and H3were shifted upfield and were diagnostic for product formation.The singlet of methyl groups on positively charged nitrogen atom(H3) were shifted from 3.39 ppm to 3.27 ppm. Similarly, methylgroups on camphor moiety H2 and H1 were moved from1.05 ppm to 1.01 ppm and 0.90 ppm to 0.85 ppm respectively.The hydrolysis of quaternary ammonium salt 1e required ca.10 h. Monitoring of hydrolysis of compounds 1f and 1g gave iden-tical signal changes in 1H NMR spectra. The only difference com-pared to salt 1e was the time needed for complete hydrolysis.Ammonium salt 1f needed ca. 8 h and salt 1g ca. 6 h for totalhydrolysis. The signals of long chain alcohol (e.g. H4) completelydisappeared from the 1H NMR spectra once the hydrolysis wascompleted due to a very low solubility. A low solubility of com-pounds 2 in D2O did not allowed us to prepare solutions suitablefor experiments. We just can predict, according to the structureof ammonium salts 2 that analogues 2a–2d stay stable under

physiological conditions similar to salts 1a–1d as well as ‘‘soft’’salts 2e–2f undergo hydrolysis like 1e–1g.

4. Conclusions

In this work 14 new optically active quaternary ammoniumsalts bearing camphor derived sulfonamide moiety were success-fully synthesized and characterized. The biological activities of pre-pared compounds, differing in the nature of the hydrocarbon sidechain and the spacer linking the ammonium head to camphor moi-ety were measured and compared with clinically used benzalkoni-um bromide. The highest biological activity from all of the studiedsalts was observed for compound 1c. This salt was approximately25 times more active against S. aureus and C. albicans and 100times more active against E. coli than BAB. Moreover, almost allthe synthesized quaternary ammonium salts showed importantantimicrobial and antifungal activities. The ‘‘soft’’ hydrolysableester parts of the compounds 1e-1g and 2e-2g, respectively, didnot negatively influence the antimicrobial activity of such com-pounds. The aggregation properties were studied by tensiometry.Critical micelle concentration (CMC) and surface tension at CMC(cCMC) were calculated for each compound where it was found abreak in the plot of logarithm of surfactant‘s concentration versussurface tension. The stability 1H NMR experiments at physiologicaland acidic pH showed good stability for QUATs 1a–1d. The ‘‘soft’’analogues 1e–1g were proved to be stable under acidic conditionsbut hydrolyzed at physiological pH during 6–10 h.

Acknowledgements

Financial support of this work by the Slovak Research andDevelopment Agency under the contract No APVV-0516-12 isgratefully acknowledged by the authors.

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