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

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Synthesis, surface and antimicrobial properties of some quaternary ammonium homochiral camphor sulfonamides 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, Slovakia Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Slovakia c NMR Laboratory, Faculty of Pharmacy, Comenius University in Bratislava, Slovakia b

a r t i c l e

i n f o

Article history: Received 26 May 2014 Received in revised form 15 August 2014 Accepted 30 August 2014 Available online 10 September 2014 Keywords: Quaternary ammonium salts Antimicrobial activity Homochiral camphorsulfonamides Critical micelle concentration

a b s t r a c t A group of homochiral quaternary ammonium sulfonamides bearing hydrophobic camphor derived moieties were synthesized and characterized. The described synthetic procedure is quick and efficient. The novel quaternary ammonium bromides were tested as antimicrobial and antifungal agents. They exhibited strong antimicrobial and also antifungal activity, especially N-{2-[((1S, 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfonamido] ethyl}-N,N-dimethyltetradecan-1-aminium bromide 1c. The surface properties of prepared compounds were evaluated by surface tension measurements and critical micelle concentration (CMC) with surface tension at CMC (cCMC) was calculated. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Since the quaternary ammonium salts (QUATs) were prepared for the first time by Menschutkin (1890) in the reaction of tertiary amine with alkyl halides they had been studied during the 20th century thoroughly. Practical applications of quaternary ammonium salts had been found in textile finishes (excellent fabric softeners), antielectrostatic agents and wood preservatives (Gilbert and Moore, 2005; Kim and Sun, 2002), catalysts (Shirakawa et al., 2012; Brak and Jacobsen 2013; Bilé et al., 2012) and since 1998 also as ionic 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 mediating DNA transfection has increased (Sajomsang et al., 2013; de las Cuevas et al., 2012; Cortesi et al., 2012). The strong bactericidal activity of QUATs with long alkyl chains have been known from 1915 (Jacobs and Heidelberger, 1915) and studied further on a broad range of microorganisms such as bacteria (both G+ and G ) and fungi (Merianos 1991; Lukácˇ 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

⇑ Corresponding author at: Comenius University in Bratislava, Faculty of Pharmacy, Department of Chemical Theory of Drugs, Odbojárov 10, 83232 Bratislava, Slovakia. Tel.: +421 250117323. E-mail address: [email protected] (R. Mikláš). http://dx.doi.org/10.1016/j.ejps.2014.08.013 0928-0987/Ó 2014 Elsevier B.V. All rights reserved.

in microorganisms toward disinfectants or antibiotics (Heinzel, 1988; Morente et al., 2013) brings the necessity to supply recently applied antimicrobial agents by new, potent and safe ones and thus search for new and effective molecules goes on (Feder-Kubis and Tomczuk, 2013; Hoque et al., 2012; Semenov et al., 2011; Colomer et al., 2011; Chanawanno et al., 2010). The QUATs, with one long alkyl chain at least, belong to amphiphilic compounds. These salts possess properties such as reduction of surface tension an also the attraction for negatively charged bacteria surface. Having the ability to intercalate into phospholipid membranes they may affect the processes in biological systems inducing cell autolysis leading to the leakage of intercellular materials into the environment and cell death (Kopecká-Leitmanová et al., 1989; Devinsky et al., 1987; Mlynarcˇík et al., 1981). The mode of action of cationic surfactants on bacteria’s membrane strongly depends on ability to form micelles and intercalate into the bacterial membrane because the disruption of membrane and subsequent solubilization of its parts plays a crucial role in the cell death process. The antimicrobial effect of QUATs is bilinearly related to their micellization properties which are linearly related to the length of the alkyl chains. The antimicrobial activity for Gram-positive bacteria is optimal when the maximum of the carbon chain length is C12–C14 while for Gramnegative bacteria activity is increased with the chain length of C14–C16 (Thebault et al., 2009; Pernak and Skrzypczak, 1996; Devinsky et al., 1990, 1991). The highest antimicrobial activity shows QUATs with critical micelle concentration range 1
10 2 to 1
10 4 mol L 1 (Devínsky et al., 1985). Molecules with n-alkyl

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chain length below C4 and above C18 are antimicrobially ineffective. In the series of structurally related QUATs the antimicrobial activity increases with the growing chain length until the maximum. From this point with continuous growing chain length the antimicrobial activity starts to decrease. This phenomenon, called cut-off effect, is typical for many of the biologically active compounds and it can be caused by many reasons (limited aqueous solubility, kinetic effects, interaction with lipid bilayers or proteins) (Balgavy´ and Devínsky, 1996). The antimicrobial activity in fact is affected not only by alkyl chain length but also by other hydrophobic groups in the molecule. The study of this effect on antimicrobial properties could help in development of new active QUATs. In addition, introduction of new structural motives such as heteroatoms or aromatics (Semenov et al., 2011; Pernak et al., 2001) may result in potential antimicrobial agents with higher biological activities and they could even conquer the growing resistance phenomenon. The well-known antibacterial effect of essential oils containing bicyclical camphor or borneol (Ruiz-Navajas et al., 2012; Miguel et al., 2011) brought us to the idea to design and synthesize QUATs bearing hydrophobic camphor derived sulfonamides, hoping that incorporation of two important antimicrobially active structures in one compound will improve their bioactivity. The introduction of an ester group into the molecule contributes to better biodegradability of such compounds and in fact compounds 1e–1g, and 2e–2g belong to the so called ‘‘soft’’ quaternary ammonium type antimicrobials (Bodor et al., 1980; Bodor and Kaminski 1980). Moreover we strongly believe that looking for new highly effective antimicrobials is very important especially nowadays when new highly resistant bacterial stems are emerging (Buffet-Bataillon et al., 2012). In this study we have prepared as illustrated in Fig. 1 series of 14 new optically active quaternary ammonium salts with camphor sulfonamides and tested their antimicrobial activity against Gramnegative Escherichia coli, Gram-positive human pathogenic bacteria Staphylococcus aureus and human fungal pathogen Candida albicans. Their surface properties were studied by surface tension measurements. The critical micelle concentration was calculated from the plot of logarithm of the surfactant‘s concentration vs. surface tension.

ethyl acetate, DMSO, bromoalkanes) are commercially available. DCM was pre-dried over CaCl2 and then distilled from CaH2 under nitrogen atmosphere. Diethyl ether was distilled from sodium prior to use. Bromoacetic acid esters were synthesized by modification of already published procedure (Nguyen et al., 2004). A corresponding alcohol and bromoacetic acid were heated in the presence of toluene-4-sulfonic acid in toluene with an azeotropic removal of water. 1 H and 13C NMR spectra were measured on a Varian Gemini 300 spectrometer at 300 MHz and 75 MHz respectively. Chemical shifts have been reported in ppm relative to an internal reference (TMS). IR spectra (in KBr pellets) were recorded on FTIR Impact 400D Nicolet instrument. Polarimetric measurements were obtained using a Jasco P-1010 polarimeter at 589 nm. Elemental analyses were carried out on a Carlo Erba 1108A instrument. HR-MS analyses were made in high-resolution system LTQ Orbitrap XL (Thermo Fisher Scientific, San Jose, CA, USA). Samples were ionized by elektrospray ION MAX-ESI in positive mode, respectively. Conditions for positive ionization were: spray voltage 4 kV, sheath gas 5 arbitrary units, capillary temperature 280 °C, capillary voltage 30 V, lens voltage 90 V. Data were scanned during 1 min. in fullscan mode 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 external standard and samples were dosed by infusion system at a flow rate of 2,5 ll/min. All melting points reported were uncorrected and measured on Kofler hot stage. The surface tension measurements were performed on Krüss processor tensiometer K100 (Wilhelmy plate method). Temperature was kept constant at the desired level using thermostatted (Thermo Haake SC100) water bath. Double-distilled water was used for the preparation of all samples. Measurements of equilibrium surface tension were taken repeatedly until the change in surface tension was less than 0.08 mN m 1. The values of surface tension decrease with increasing concentration and the break point provides the CMC value and surface tension at CMC (cCMC). 2.2. Microbiology

2. Experimental procedure 2.1. Materials and methods All compounds used ((1S)-(+)-camphor-10-sulfonic acid (CSA), thionylchloride, diethyl ether, acetone, dichloromethane (DCM),

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

Fig. 1. Synthesis of QUATs derived from (1S)-(+)-camphor-10-sulfonic acid.

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

fungi had a concentration of 5  107 cfu mL 1 of bacteria and 5  105 cfu mL 1 of Candida. Concentration of microorganisms were determined spectrophotometrically at 540 nm and adjusted to absorbance A = 0.35. The microorganism suspension was added to solutions containing the tested compound and to double concentrated peptone broth medium (8%) for bacteria or Sabouraud medium (12%) for Candida. The stock solution of tested compounds was serially diluted by half. The cultures were done in 96-well microliter plates. The microorganisms were incubated for 24 h at 37 °C and then from each well 5 microliters of suspension were cultured on blood agar (bacteria) or on Sabouraud agar (fungi). After 24 h at 37 °C the lowest concentration of QUATs which prevented colony formation was determined as minimal inhibitory concentration (MIC). (Lukácˇ et al., 2010) As a standard benzalkonium bromide (BAB, AjatinÒ) was used. 2.3. Synthesis Enantiopure camphor sulfonylchloride 5 was prepared according to the known procedure (Gayet et al., 2004). 2.3.1. N-(2-dimethylaminoethyl)-1-[(1S)-7,7-dimethyl-2oxobicyclo[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 solution of 5 (10 g, 0.04 mol) in anhydrous DCM (30 ml) at 0 °C during 30 min. After the addition was complete the reaction mixture was heated subsequently to reflux for 2 h. and then mixed over night at ambient temperature. The solvent was removed on rotary evaporator and 50 ml of dry diethyl ether was added to the residue. The resulting suspension was mixed 30 min and filtered. A white powder of 3.HCl thus obtained was washed twice with 25 ml of dry diethyl ether and crystallized from acetone/methanol (10:1, v/v). A 3.HCl was prepared in 90% yield. Free base 3 was eliberated by dropwise addition of 10% aqueous NaOH into the suspension of 3.HCl in ethyl acetate at 0 °C until the pH was adjusted to 12. Aqueous layer was separated and washed 3 times with 30 ml of ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4 and evaporated to yield yellowish oil (97%). The product was used in the next reaction without further purification. 1H NMR (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-2oxobicyclo[2.1.1]heptan-1-yl]methane sulfonamide (4) Compound 4 was prepared by the same procedure described for 3 by the reaction of N-methylpiperazine (4.96 g, 0.05 mol) with sulfonylchloride 5 (12.4 g, 0.05 mol) in DCM. Sulfonamide 4 was isolated 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 1 and 2 10 mmol of sulfonamide 3 or 4 were mixed with 1.3 equivalent of alkylating bromoderivative in CH3CN (25 ml). Reaction mixture was stirred at ambient temperature for 2 h, then refluxed for 16 h (series a–d) or 8 h (series e–g) respectively and cooled. The reaction mixture was placed in the freezer overnight and white crystals were filtered off, washed twice with 25 ml of anhydrous

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diethyl ether and the crude product was recrystallized repeatedly from anhydrous acetone-methanol mixture. 2.3.4. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-N,Ndimethyldecan-1-aminium bromide (1a) Yield 76%, colorless crystals, mp 115–117 °C. [a]20 D = +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. Elemental 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-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-N,Ndimethyldodecan-1-aminium bromide (1b) Yield 68%, colorless crystals, mp 98–101 °C. [a]20 D = +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, N 5.08, S 5.81. Found: C 56.06, H 9.03, N 4.80, S 6.41. HRMS for C26H51N2O3S (M + H)+, calcd 471.3615, found 471.3606. 2.3.6. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfonamido]ethyl}-N,Ndimethyltetradecan-1-aminium bromide (1c) Yield 65%, colorless crystals, mp 136–138 °C. [a]20 D = +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: C 56.80, H 9.28, N 4.40, S 6.11. HRMS for C28H55N2O3S (M + H)+, calcd 499.3928, found 499.3919. 2.3.7. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-N,Ndimethylhexadecan-1-aminium bromide (1d) Yield 74%, colorless crystals, mp 105–106 °C. [a]20 D = +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:

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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-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfonamido]ethyl}-N,Ndimethyl-2-oxoethanaminium bromide (1e) Yield 52%, colorless crystals, mp 134–138 °C. [a]20 D = +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. for C26H49BrN2O5S: C 53.69, H 8.49, N 4.82, S 5.51. Found: C 51.86, H 8.15, N 4.24, S 5.80. HRMS for C26H49N2O5S (M + H)+, calcd 501.3357, found 501.3349. 2.3.9. Quaternary salt N-{2-[((1S, 4R)-7,7-dimethyl-2oxobicyclo[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]20 D = +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-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]ethyl}-N,Ndimethyl-2-oxo-2-(tetradecyloxy)ethanaminium bromide (1g) Yield 78%, colorless crystals, mp 133–134 °C. [a]20 D = +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. Elemental 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-2oxobicyclo[2.2.1]heptan-1-yl)methyl sulfonamido]-1methylpiperazinium bromide (2a) Yield 71%, colorless crystals, mp 225–226 °C. [a]20 D = +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. Elemental Anal. 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-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-dodecyl-1methylpiperazinium bromide (2b) Yield 53%, colorless crystals, mp 228–229 °C. [a]20 D = +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. IR m/cm 1 2924, 2853, 1748, 1632, 1474, 1352, 1329, 1155, 1072, 771. Elemental Anal. Calcd. for C27H51BrN2O3S: C 57.53, H 9.12, N 4.97, S 5.69. Found: C 56.37, H 8.85, N 4.67, S 6.63. HRMS for C27H51N2O3S (M + H)+, calcd 483.3615, found 483.3609. 2.3.13. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-methyl-1tetradecylpiperazinium bromide (2c) Yield 48%, colorless crystals, mp 222–225 °C. [a]20 D = +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. IR m/cm 1 2923, 2853, 1747, 1632, 1468, 1350, 1331, 1155, 1068, 1049, 779. Elemental Anal. Calcd. for C29H55BrN2O3S: C 58.86, H 9.37, N 4.73, S 5.42. Found: C 57.65, H 9.02, N 4.57, S 5.89. HRMS for C29H55N2O3S (M + H)+, calcd 511.3928, found 511.3919. 2.3.14. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-hexadecyl1-methylpiperazinium bromide (2d) Yield 53%, colorless crystals, mp 221–224 °C. [a]20 D = + 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, H 9.60, N 4.52, S 5.17. Found: C 59.15, H 9.45, N 4.39, S 5.59. HRMS for 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-1yl)methylsulfonamido]-1-methylpiperazinium bromide (2e) Yield 72%, colorless crystals, mp 186–189 °C. [a]20 D = +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

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. for C27H49BrN2O5S: C 54.63, H 8.32, N 4.72, S 5.40. Found: C 53.47, H 8.12, N 4.39, S 6.13. HRMS for C27H49N2O5S (M + H)+, calcd 513.3357, found 513.3347. 2.3.16. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1(dodecyloxycarbonylmethyl)-1-methylpiperazinium bromide (2f) Yield 69%, colorless crystals, mp 179–181 °C. [a]20 D = +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, N 4.51, S 5.16. Found: C 54.98, H 8.42, N 4.27, S 5.73. HRMS for C29H53N2O5S (M + H)+, calcd 541.3670, found 541.3660. 2.3.17. Quaternary salt 4-[((1S, 4R)-7,7-dimethyl-2oxobicyclo[2.2.1]heptan-1-yl)methylsulfon amido]-1-methyl-1(tetradecyloxycarbonylmethyl)piperazinium bromide (2g) Yield 79%, colorless crystals, mp 174–176 °C. [a]20 D = +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. for C31H57BrN2O5S: C 57.30, H 8.84, N 4.31, S 4.93. Found: C 56.28, H 8.60, N 3.87, S 5.52. HRMS for C31H57N2O5S (M + H)+, calcd 569.3983, 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 hydrolysis was monitored by 1H NMR. Spectra were taken at the given period of time for the samples kept at 37 °C. PBS buffer, pH 7.2 was 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 mixing of KH2PO4 (0.131 g) in D2O (5 ml) and adjusted to pH 2.5 by dropwise addition of 10% HCl in H2O. 3. Results and discussion Enantiopure quaternary ammonium salts 1 and 2 were synthesized as illustrated in Fig. 1. starting from (1S)-camphor-10-sulfonic acid. Although sulfonylchloride 5 is commercially available as a starting material, (1S)-camphor-10-sulfonic acid proved to be less expensive and can be easily converted to the sulfonylchloride 5 by the procedure already published (Gayet et al., 2004). Thus (1S)-camphor-10-sulfonic acid was reacted with thionylchloride providing compound 5 in 86% yield after crystallization from petroleum ether. The preparation of camphor sulfonamides 3 and 4 was carried out by drop wise addition of equimolar amount of 5 in anhydrous DCM into the solution of N,N-dimethylethan-1,2diamine and N-methylpiperazine in DCM respectively. The

33

presence of tertiary amino group in the structure allowed us to omit the use of another base for binding hydrochloric acid formed during the reaction. The hydrochlorides 3.HCl and 4.HCl thus obtained were purified by crystallization. The free bases 3 and 4 were eliberated by treatment with aqueous NaOH and quaternized by appropriate bromoderivative in acetonitrile in order to give two series of QUATs 1 and 2. All quaternary ammonium sulfonamides were obtained as colorless crystals after several crystallizations from anhydrous acetone–methanol mixture in yields ranged from 48 to 79%. They were identified and characterized thoroughly from spectral and analytical data. The higher differences in elemental analysis, mainly for carbon, are caused by hydrophilic nature of prepared salts. Approximately 0.5–1 molecule of water per QUAT‘s molecule is present in the sample. HR-MS spectra clearly proved the structure of synthesized QUATs. Quaternary ammonium salts exhibit strong antimicrobial activities and they are widely used as a disinfectants and antiseptics. The main target site of QUATs is the cytoplasmic membrane surrounding the cytoplasm of a cell and comprised of a phospholipid bilayer. QUATs are able to intercalate into phospholipid bilayer which is accompanied by membrane disorganization and structural and functional changes in the cell wall inducing leakage of intracellular components (Tischer et al., 2012; Hoque et al., 2012; Wessels and Ingmer, 2013; Gilbert and Moore, 2005). In addition QUATs were found to inhibit ATP synthesis by neutralizing the proton motive force (PMF) (Denyer and Hugo, 1977). The PMF is initiated by a proton gradient across the cytoplasmic membrane and it is involved in many respiratory and photosynthetic processes including ATP synthesis. Quaternary ammonium salts are surface active agents and therefore, they denature proteins anchored in the cytoplasmic membrane or cause dissociation of an enzyme from its prosthetic group. This effect was observed at concentrations much higher than lethal ones so the enzyme inhibition is not the primary or main lesion caused by cationic surfactants (Merianos 1991). It has been shown that some bisammonium salts have intracellular target and bind to DNA which leads to the inhibition of DNA replication (Zinchenko et al., 2004; Menzel et al., 2011). On the other hand, for most of the QUATs no specific target site has been recognized. However it is not excluded that there can exist some target specificities as shown by Menzel et al. (2011) and Zhang et al. (2013) because the antimicrobial activity of QUATs fluctuates significantly against various types of microorganisms and explanation simply by the cationic charge and hydrophobic tail cannot be used. The antimicrobial activities of the sulfonamide QUATs, were determined as a minimal inhibitory concentration (MIC, [lmol l 1]) against the Gram-positive human pathogenic bacteria S. Aureus, Gram-negative bacteria E. Coli and human fungal pathogen C. Albicans, the values for which are given in Table 1. The MIC values were determined as lowest concentration of QUATs that completely prevented visible colony formation. All the studies were carried out in DMSO. In order to prove that the solvent does not influence bacterial and fungal growth a test with pure solvent was performed. This control test detected no inhibiting activity. Clinically used benzalkonium bromide (BAB, AjatinÒ) was used as a standard. Most of the prepared QUATs, except of 1a, 1e, 2a and 2g, showed higher antibacterial activity against Gram-positive bacteria S. aureus than standard BAB. The similar trend was observed also in the activity against E. coli. The only compounds with lower activity against E. coli compared to BAB were 1a, 2d and 2g. In the case of C. albicans it was 1a, 1b, and 2a–2d. Activities of investigated compounds were slightly higher for Gram-positive bacteria than for Gram-negative bacteria except of compound 1e which exhibits the same antimicrobial activity for both Gram-positive and Gram-negative bacteria. Surprisingly, the activity of soft salts 1e–1g and 2e–2g against fungi was mainly similar or higher to that

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

Table 1 MIC values (lmol l

in various biological and toxic activities in practically every amphiphile homologous series tested. Several theories have been proposed to explain the cut-off effect. Ferguson (1939) was one of the first who has suggested that the cut-off effect in biological activity could be caused by a decrease in the achievable compound concentration at the site of action due to its limited solubility. According to his model, the drug partition coefficient between the aqueous solution and the site of action increases less rapidly with the increasing chain length than the aqueous solubility decreases. Thus the compounds with longer hydrophobic chains have limited partitioning which results in significantly lower concentration at the site of action with no or small biological effect. Several authors (Ross et al., 1953; Birnie et al., 2000) have proposed that micellization is responsible for cut-off effect in biological activities of surfactant‘s type biocides. Also this theory is based on decreased concentration of active compound at the site of action but due to the fact that the long chain surfactant molecules have higher predisposition for micellization than the tendency to move toward the cell membrane (interface). From our previous studies (Balgavy´ and Devínsky, 1996) we have proposed the free volume hypothesis to explain the cut-off effect. In the bilayer, the amphiphile polar groups will interact with lipid polar groups and their chains will orient parallel to the lipid hydrocarbon chains. At this location, the packing density of the bilayer hydrophobic region must be influenced due to lateral expansion of the bilayer and formation of free volume close to the amphiphile chain

1

) for prepared QUATs.

Compound

S. aureus ATCC 6538

E. coli CNCTC 377/ 79

C. albicans CCM 8186

1a 1b 1c 1d 1e 1f 1g 2a 2b 2c 2d 2e 2f 2g BAB

149.20 8.85 1.05 2.01 33.57 8.02 7.67 45.60 10.60 5.20 9.80 10.28 5.63 37.55 26.00

298.41 17.69 2.2 128.54 33.57 16.04 61.40 182.20 173.10 41.20 1260.40 82.20 78.40 480.90 260.00

596.82 35.40 1.05 4.01 16.78 8.02 1.92 182.2 86.60 164.90 315.10 20.55 2.45 9.39 26.00

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

6,0

7,0 6,5

5,5

log (1/MIC)

log (1/MIC)

6,0 5,0 4,5 4,0

5,5 5,0 4,5 4,0 3,5

3,5 3,0 3,0

2,5 10

11

12

13

14

15

10

16

11

12

13

14

15

16

n

n

Fig. 2. Relation between the number of carbon atoms in alkyl chain (n) and antimicrobial activity of 1a–1d (left) and 2a–2d (right). S. aureus j E. colid C. albicans N.

5,8

5,6 5,4

5,6

5,0

5,2

4,8

log (1/MIC)

log (1/MIC)

5,2 5,4

5,0 4,8 4,6

4,6 4,4 4,2 4,0 3,8

4,4

3,6 3,4

4,2

3,2 10

11

12

n

13

14

10

11

12

13

14

n

Fig. 3. Relation between the number of carbon atoms in alkyl chain (n) and antimicrobial activity of 1e–1g (left) and 2e–2g (right), CH2COO group is not counted in n. S. aureus j E. colid C. albicans N.

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

ends. The bigger is the difference between the chain length of phospholipids and amphiphile the bigger free volume is created. The free volume can be eliminated by rearrangement of hydrocarbon chains (increased frequency of trans–gauche isomerization, bending and chain interdigitation) which results in the change of the bilayer thickness. The interaction of compounds with a short hydrophobic chain creates a large free volume but the number of molecules built in the membrane is too small so the total free volume created in the bilayer hydrophobic region will be small. As the amphiphile chain becomes long and comparable with lipid hydrocarbon chains the free volume will be small. This will result in small changes in the membrane structure and low biological response. Amphiphilic molecules with chain length between these extremes will thus induce maximal free volume and therefore they will cause maximal membrane disruption accompanied by maximal biological response. We should like to note that biological systems are complicated and it is not excluded that the cut-off effect in these systems could be caused by a combination of different mechanisms.

Table 2 Cmc and ccmc values of quaternary ammonium salts 1a–1g at 25 °C. 3

Compound

cmc (mold m

1a 1b 1c 1d 1e 1f 1g

4.39 ± 0.40  10 2.62 ± 0.12  10 1.31 ± 0.01  10 0.81 ± 0.06  10 1.91 ± 0.04  10 0.72 ± 0.14  10 2.71 ± 0.24  10

ccmc (mN m 1)

) 3

29.5 34.3 36.8 39.3 32.3 32.5 24.9

3 3 5 3 3 4

The highest inhibition activity of hard salts 1a–1d and 2a–2d against Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus was found for compounds with fourteen carbon atoms in alkyl chain. The behavior of fungi C. albicans was not so predictable. In the series of compounds 1a–1d the highest fungicidal activity was also observed for compounds with fourteen carbon atoms in alkyl chain. The piperazine derived salts 2a–2d exhibited the highest activity with their alkyl chain length being twelve carbon atoms only. The series of soft quaternary salts 1e–1g and 2e– 2g exhibit the best antimicrobial activity against Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli with twelve carbon atoms in the long alkyl chain (CH2COO group is not counted). The best inhibition of C. albicans growth by soft salts was also determined being twelve carbon atoms in alkyl chain for piperazine derivatives 2e–2g but fourteen carbon atoms for salts 1e-1g. The ‘‘soft’’ hydrolysable ester parts of the compounds 1e–1g and 2e–2g, respectively, did not negatively influence the antimicrobial activity of such compounds. The surface properties of prepared QUATs were investigated by surface tension measurements. Quaternary ammonium salts 2a–2g exhibit low solubility in water. Thus only linear decreasing of surface tension was observed from the plots. To increase the solubility and thus the desired concentration of compounds 2a–2g we have also examined surface behavior at increased temperature (up to 41 °C) but with no break in the plot. The determination of critical micelle concentration of compounds 1a–1g with more flexible ethylene spacer between camphor moiety and polar head was more successful. Critical micelle concentrations of QUATs are shown in Table 2. The plots of surface tension against the logarithm of surfactant‘s concentration are presented in Fig. 4. It can be seen form Table 2 that values of the critical micelle concentration

65

55

-1

55 50 45 40 35 30

1e 1f 1g

50 45 40 35 30 25

25 -5,5

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

-5,0

-4,5

log c

-4,0

-3,5

log c 65

-1 Surface tension (mNm )

-1 Surface tension (mNm )

Surface tension (mNm )

1a 1d

60

1b 1c

60 55 50 45 40 35 30 -3,8

-3,6

-3,4

-3,2

-3,0

-2,8

-2,6

-2,4

-2,2

-2,0

-1,8

log c Fig. 4. Surface tension versus the logarithm of the aqueous molar concentration of 1a–1g at 25 °C.

-3,0

-2,5

-2,0

36

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

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

physiological conditions similar to salts 1a–1d as well as ‘‘soft’’ salts 2e–2f undergo hydrolysis like 1e–1g. 4. Conclusions

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

decrease with number of carbon atoms in the chain being increased in both of the series 1a–1d and 1e–1g respectively. To study the stability of prepared QUATs under physiological and acidic conditions 1H NMR technique was used. Quaternary ammonium salts 1 were subjected to hydrolysis (Fig. 5) in PBS/ D2O buffer (pH 7.2) and PBS/HCl buffer (pH 2.5). The reactions were performed at 37 °C in NMR tubes. As we expected, the salts 1a–1d without an ester functional group, did not undergo hydrolysis under studied conditions and NMR spectra showed no changes after 480 h. The ‘‘soft’’ analogues 1e–1g with hydrolysable ester part surprisingly showed good stability at pH 2.5 with no modification in NMR spectra after 480 h. As illustrated in Fig. 6, exposure of ammonium salt 1e to physiological pH 7.2 showed changes in NMR spectra almost instantly. The signals of protons H1, H2 and H3 were 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, methyl groups on camphor moiety H2 and H1 were moved from 1.05 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 identical signal changes in 1H NMR spectra. The only difference compared to salt 1e was the time needed for complete hydrolysis. Ammonium salt 1f needed ca. 8 h and salt 1g ca. 6 h for total hydrolysis. The signals of long chain alcohol (e.g. H4) completely disappeared from the 1H NMR spectra once the hydrolysis was completed due to a very low solubility. A low solubility of compounds 2 in D2O did not allowed us to prepare solutions suitable for experiments. We just can predict, according to the structure of ammonium salts 2 that analogues 2a–2d stay stable under

In this work 14 new optically active quaternary ammonium salts bearing camphor derived sulfonamide moiety were successfully synthesized and characterized. The biological activities of prepared compounds, differing in the nature of the hydrocarbon side chain and the spacer linking the ammonium head to camphor moiety were measured and compared with clinically used benzalkonium bromide. The highest biological activity from all of the studied salts was observed for compound 1c. This salt was approximately 25 times more active against S. aureus and C. albicans and 100 times more active against E. coli than BAB. Moreover, almost all the synthesized quaternary ammonium salts showed important antimicrobial and antifungal activities. The ‘‘soft’’ hydrolysable ester parts of the compounds 1e-1g and 2e-2g, respectively, did not negatively influence the antimicrobial activity of such compounds. 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 a break in the plot of logarithm of surfactant‘s concentration versus surface tension. The stability 1H NMR experiments at physiological and acidic pH showed good stability for QUATs 1a–1d. The ‘‘soft’’ analogues 1e–1g were proved to be stable under acidic conditions but hydrolyzed at physiological pH during 6–10 h. Acknowledgements Financial support of this work by the Slovak Research and Development Agency under the contract No APVV-0516-12 is gratefully acknowledged by the authors. References Balgavy´, P., Devínsky, F., 1996. Cutt-off effects in biological activities of surfactants. Adv. Colloid Interf. Sci. 66, 23–63, http://dx.doi.org/10.1016/00018686(96)00295-3. Bilé, E.G., Cortelazzo-Polisini, E., Denicourt-Nowocki, A., Sassine, R., Launay, F., Roucoux, A., 2012. Chiral ammonium-capped rhodium(0) nanocatalysts: synthesis, characterization, and advances in asymmetric hydrogenation in neat water. ChemSusChem 5, 91–101. http://dx.doi.org/10.1002/ cssc.201100364. Birnie, C.R., Malamud, D., Schnaare, R.L., 2000. Antimicrobial evaluation of N-alkyl betaines and N-alkyl-N, N-dimethylamine oxides with variations in chain length. Antimicrob. Agents Chemother. 44, 2514–2517. http://dx.doi.org/ 10.1128/AAC.44.9.2514-2517.2000. Bodor, N., Kaminski, J.J., 1980. Soft drugs. 2. Soft alkylating compounds as potential antitumor agents. J. Med. Chem. 23, 566–569. http://dx.doi.org/10.1021/ jm00179a018. Bodor, N., Kaminski, J.J., Selk, S., 1980. Soft drugs. 1. Labile quaternary ammonium salts as soft antimicrobials. J. Med. Chem. 23, 469–474. http://dx.doi.org/ 10.1021/jm00179a001. Brak, K., Jacobsen, E.N., 2013. Asymmetric ion-pairing catalysis. Angew. Chem. Int. Ed. 52 (2), 534–561. http://dx.doi.org/10.1002/anie.201205449.

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