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Synthesis and antimicrobial properties of camphorsulfonic acid derived imidazolium salts

Synthesis and antimicrobial properties of camphorsulfonic acid derived imidazolium salts Keywords Kúcové slová: imidazolium salts, antimicrobial activity, camphorsulfonic acid imidazóliové soli, antimikróbna aktivita, kyselina gáforsulfónová INTRODUCTION The strong bactericidal activity of quaternary ammonium salts (QAS) with long alkyl chains have been known since 1915 (Jacobs & Heidelberger, 1915) and studied further on a broad range of microorganisms such as Gram positive (G+) and Gram negative (G-) bacteria and fungi (Lukác et al., 2010; Miklás et al., 2012, 2014), certain viruses (Wong et al., 2002), herbicidal salts (Cojocaru et al., 2013) and even anticancer agents (Kaushik et al., 2012). 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 (Devínsky et al., 1987; Mlynarcík et al., 1981). The widespread importance of surfactants in practical applications (Brak & Jacobsen, 2013; Cortesi et al., 2012; Gilbert & Moore, 2005; Zhi et al., 2012) and difficulties to recover or reuse them due to their water solubil* miklas@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae ity causes their accumulation in environment, thus affecting aquatic ecosystem. Most QASs are not easily biodegraded; therefore, the aquatic microorganisms remain in contact with them for a longer period of time, which increases their toxicity. It was found (Kümmerer et al., 1997) that LC50 of benzalkonium chloride to fish was between 0.5 and 5.0 mg l-1 and to daphnids even from 0.1 to 1.0 mg l-1. In addition, the presence of QASs may decrease the biodegradation efficiency of linear alkylbenzene sulfonates. The toxicity of QASs depends on both polar and hydrophobic parts of the molecule. Generally, the double-tail surfactants are less toxic than the single-tail counterparts (Pinnaduwage et al., 1989). The length of the hydrophobic alkyl chain in QASs plays also an important role for toxicity. While for aliphatic single-tail cationic surfactants, the toxicity increases with the increasing alkyl chain length; (Rasia et al., 2007) for Acetone was distilled from potassium carbonate. Bromoacetic double-tail cationic surfactant, the toxicity decreases as the acid esters (Michniak et al., 1996) and amides (Hoque et al., alkyl chain length increases (Spelios & Savva, 2008). Concern- 2012) were synthesized by modification of published proing the polar part of the QAS`s molecule, the positive charge cedures. 1H and 13C NMR spectra were measured on a Varian can be located on a quaternary ammonium group or delo- Gemini 300 spectrometer at 300 MHz and 75 MHz, respectively. calized in heterocyclic ring or in a guanidine group. Delocali- Chemical shifts have been reported in ppm relative to an interzation of the positive charge mostly helps to decrease the nal reference (TMS). IR spectra (in KBr pellets) were recorded toxicity of such surfactants (Coleman et al., 2012). In order to on FTIR Impact 400D Nicolet instrument. Polarimetric measavoid systemic toxicity and environmental persistency of QAS urements were obtained using a Jasco P-1010 polarimeter at antimicrobials, the "soft" drug approach developed by Bodor 589 nm. Elemental analyses were carried out on a Carlo Erba et al. (1980) was applied to this problem. The structural "soft" 1108A instrument. All melting points reported were uncorrectanalogues, compared to their "hard" counterparts, have a spe- ed and measured on Kofler hot stage. cific easily degradable functional group built into their structures to provide their one-step detoxification. Various "soft" 2.2 Microbiology surfactants possessing a cleavable moiety, such as an ester The antimicrobial activity was tested against Gram-negative and/or amide bond, have been synthesized and their antimi- bacteria Escherichia coli CNCTC 377/79, Gram-positive baccrobial activity and biodegradation was tested (Colomer et al., teria Staphylococcus aureus ATCC 6538 and fungi Candida al2012; Devínsky et al., 1991; Fan et al., 2013). The well-known bicans CCM 8186. Solutions of the compounds studied were antibacterial effect of essential oils containing bicyclic cam- prepared in DMSO (5%, w/v). A suspension of the standard phor or borneol (avar et al., 2012; Ruiz-Navajas et al., 2012) microorganism, prepared from 24-h cultures of bacteria in resulted in the idea to design and synthesize potentially "soft" blood agar and from 24-h cultures in the Sabouraud agar for QASs bearing hydrophobic chiral camphor derived moieties, fungi had a concentration of 5x107 cfu ml-1 of bacteria and hoping that incorporation of two important antimicrobial ac- 5x105 cfu ml-1 of Candida. Concentration of microorganisms tive structures in one compound will improve their bioactivity. were determined spectrophotometrically at 540 nm and adIn this study, we have prepared a series of six new optically justed to absorbance A = 0.35. The microorganism suspenactive quaternary imidazolium salts, which could be specified sion (5 l) was added to solutions containing the compound as potentially "soft" disinfectants due to the ester or amide under examination (100 l) and to double-concentrated pepbonds in the structure (Fig. 1). Their antimicrobial activity was tone broth medium (8%) for bacteria or Sabouraud medium tested against Gram-negative Escherichia coli, Gram-positive (12%) for Candida (100 l).The double-concentrated medium human pathogenic bacteria Staphylococcus aureus and hu- was used due to addition of the same volume of solution man fungal pathogen Candida albicans. containing the compound tested, which resulted in the desired standard concentration of medium for experiments. The stock solution of tested compounds was serially diluted by 2. EXPERIMENTAL half. The cultures were carried out using 96-well microliter 2.1 Materials and methods plates. The microorganisms were incubated for 24 h at 37°C All compounds used ((1S)-(+)-camphor-10-sulfonic acid (CSA), and then from each well, 5 l of suspension were cultured on thionylchloride, triethylamine (TEA), diethyl ether, acetone, blood agar (bacteria) or on Sabouraud agar (fungi). After 24 h dichloromethane (DCM), ethyl acetate, petroleum ether (40­ at 37°C, the lowest concentration of QAS, which prevented 65°C), DMSO, bromoacetyl bromide, fatty alcohols and amines) colony formation, was determined as minimal inhibitory conare commercially available. DCM was pre-dried over CaCl2 and centration (MIC). Benzalkonium bromide (BAB, Ajatin®) and then distilled from CaH2 under a nitrogen atmosphere. Diethyl carbethopendecinium bromide (Septonex®) were used as the ether was pre-dried from KOH and then distilled from sodium. standards. H N SOCl2 DCM, TEA SO2Cl R-Br CH3CN O2 S O N 100°C O2 S O N SO3H 1a 1b 1c 2a 2b 2c N+ R R = -CH2COO-decyl R = -CH2COO-dodecyl R = -CH2COO-tetradecyl R =-CH2CONH-decyl R = -CH2CONH-dodecyl R = -CH2CONH-tetradecyl Figure 1. Preparation of camphorsulfonic acid derived imidazolium salts of the compounds in series 1 and 2. 2.3 Synthesis Enantiopure camphor sulfonylchloride 4 was prepared according to the known procedure (Gayet et al., 2004). 1-((1H-imidazol-1-ylsulfonyl)methyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one (3) To a solution of imidazole (7.1 g, 0.104 mol) and TEA (11.1 ml, 0.08 mol) in anhydrous DCM (50 ml) was added drop wise a solution of 4 (20.12 g, 0.08 mol) in anhydrous DCM (80 ml) at 0°C over 30 min. After the addition was complete, the reaction mixture was heated to reflux for 1.5 h. and then stirred overnight at ambient temperature. A precipitate was filtered off and filtrate was extracted with 20% (w/v) aqueous solution of Na2CO3 (2 x 50 ml) and brine (50 ml). The organic layer was dried over anhydrous Na2SO4 and evaporated to yield a yellowish solid. The crude product was recrystallized from acetone/petroleum ether (3:1, v/v) mixture to yield 19.1 g (84%) of white crystals. General procedure for the synthesis of quaternary salts 1 and 2 The sulfonamide 3 (12 mmol) was mixed with 1.2 equivalents of the appropriate alkylating bromoderivative in CH3CN (20 ml). Reaction mixture was stirred at ambient temperature for 2 h., then refluxed for 24 h. and allowed to cool. The reaction mixture was placed in the freezer for 2 days and the resulting crystals were filtered off, washed twice with 25 ml of anhydrous diethyl ether and the crude product was recrystallized repeatedly from anhydrous acetone. Characterisation and spectral data of the prepared salts are sumarized in Tables 1 and 2. 3. RESULTS AND DISCUSSION Enantiopure QASs of the compounds in series 1 and 2 were synthesized as illustrated in Fig. 1. starting from (1S)-camphor10-sulfonic acid. Although sulfonylchloride 4 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 4 by the published procedure (Gayet et al., 2004). Thus (1S)-camphor-10-sulfonic acid was reacted with thionylchloride providing compound 4 in 86% yield after crystallization from petroleum ether. The preparation of camphor sulfonamide 3 was carried out by dropwise addition of 4 in anhydrous DCM into the solution of imidazole in DCM in the presence of TEA as a base. Two series of imidazolium salts 1 and 2 were formed after quaternization of 3 by n-alkyl esters or N-alkyl amides of bromoacetic acid (alkyl = decyl, dodecyl, tetradecyl) in acetonitrile. All imidazolium salts were obtained as colorless crystals after several crystallizations from anhydrous acetone in yields ranging from 24 to 46%. They were identified and characterized thoroughly from spectral and analytical data. The antimicrobial activities of imidazolium salts, were determined as a MIC, [mol 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 3. The MIC values were determined as lowest concentration of the imidazolium salt that completely prevented visible colony formation. All the compounds were dissolved in DMSO for biological evaluation. 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 inhibitory activity. Clinically used benzalkonium bromide (BAB, Ajatin®) and carbethopendecinium bromide (Septonex®) were used as standards. According to the results, it can be observed that all of the synthesized imidazolium salts exhibit growth inhibition effect against all three types of microbes, with higher efficiency against S. aureus and C. albicans (Fig. 2). Gram-negative E. coli was found to be most resistant to the prepared salts, presumably due to the cell membrane composition. Gram-negative bacteria contain an outer membrane with an external com- Table 1. Characterisation of camphorsulfonic acid derived imidazolium salts of the compounds in series 1 and 2 Compound Formula / []D21 (conc., solvent) C25H41BrN2O5S/ - 0.86 (0.1 g/100 ml, CHCl3) C27H45BrN2O5S/ - 0.44 (0.1 g/100 ml, CHCl3) C29H49BrN2O5S/ - 0.34 (0.1 g/100 ml, CHCl3) C25H42BrN3O4S/ - 9.34 (0.1 g/100 ml, CHCl3) C27H46BrN3O4S/ - 6.12 (0.1 g/100 ml, CHCl3) C29H50BrN3O4S/ - 5.47 (0.1 g/100 ml, CHCl3) C13H18N2O3S/ - 15.5 (0.1 g/100 ml, CHCl3) wi(calc.)/% wi(found)/% C 53.47 53.29 55.00 54.91 56.39 56.22 53.56 53.51 55.09 54.94 56.48 56.39 55.30 55.12 H 7.36 7.43 7.69 7.84 8.00 8.06 7.55 7.39 7.88 7.83 8.17 8.24 6.43 6.51 N 4.99 5.11 4.75 4.88 4.54 4.68 7.50 7.67 7.14 7.21 6.81 6.74 9.92 9.78 S 5.71 5.58 5.44 5.20 5.19 5.02 5.72 5.91 5.45 5.69 5.20 5.44 11.36 11.49 Yield % 46 38 41 24 33 38 84 M.p. °C 153­155 141­143 137­138 134­135 123­124 113­114 173­175 1a 1b 1c 2a 2b 2c 3 Table 2. Spectroscopic data of camphorsulfonic acid derived imidazolium salts of the compounds in series 1 and 2 Compound 1a Spectral data IR, /cm-1: 3057, 2957, 2922, 2852, 1751,1563, 1467, 1234, 1183,1042, 969, 759, 601 1H NMR (DMSO-d6, 300 MHz) 10.14 (s, 1H); 8.62 (s, 1H); 7.63 (s, 1H); 5.37 (s, 2H); 4.18 (t, 2H, J =7.2 Hz); 3.48 (d, 1H, J = 14.83 Hz); 2.88 (d, 1H, J = 14.83 Hz); 2.39­2.28 (m, 2H); 2.11­1.87 (m, 5H); 1.66 (t, 2H, J = 6.87Hz); 1.26 (s, 14H); 1.05 (s, 3H); 0.9­0.83 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.1; 165.8; 139.3; 133.5; 123.2; 67.1; 58.4; 50.3; 48.1; 47.6; 43,0; 42.5; 31.8; 29.5; 29.4; 29.3; 29.2; 28.3; 27.0; 25.7; 24.5; 22.6; 19.8; 19.7; 14.1. IR, /cm-1: 3111, 2957, 2921, 2851, 1749, 1583, 1468, 1399, 1374, 1236, 1182, 1041, 968, 866, 763, 601 1 H NMR (DMSO-d6, 300 MHz) 10.13 (s, 1H); 8.61 (s, 1H); 7.63 (s, 1H); 5.36 (s, 2H); 4.18 (t, 2H, J =7,2 Hz); 3.48 (d, 1H, J = 14.84 Hz); 2.88 (d, 1H, J = 14.84 Hz); 2.39­2.28 (m, 2H); 2.11­1.87 (m, 5H); 1.66 (t, 2H, J = 6.87Hz); 1.26 (s, 18H); 1.05 (s, 3H); 0.9­0.83 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.1; 165.9; 139.3; 133.5; 123.3; 67.1; 58.4; 50.3; 48.1; 47.6; 43,0; 42.6; 31.9; 29.7; 29.6; 29.5; 29.4; 29.3; 29.2; 28.3; 27.0; 25.7; 24.6; 22.7; 19.8; 19.7; 14.1. IR, /cm-1: 3111, 2956, 2920, 2851, 1748, 1584, 1468, 1396, 1374, 1234, 1182, 1042, 966, 788, 601 1 H NMR (DMSO-d6, 300 MHz) 10.13 (s, 1H); 8.61 (s, 1H); 7.63 (s, 1H); 5.36 (s, 2H); 4.18 (t, 2H, J =7,2 Hz); 3.48 (d, 1H, J = 14.84 Hz); 2.88 (d, 1H, J = 14.84 Hz); 2.39­2.28 (m, 2H); 2.11­1.87 (m, 5H); 1.66 (t, 2H, J = 6.87Hz); 1.26 (s, 22H); 1.05 (s, 3H); 0.9­0.83 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.1; 166.0; 139.5; 133.8; 123.3; 67.1; 58.5; 50.3; 48.1; 47.7; 43.0; 42.6; 31.9; 29.7(2C); 29.6; 29.5; 29.4; 29.2; 28.4; 27.0; 25.7; 24.6; 22.7; 19.8; 19.7; 14.1. IR, /cm-1: 3447, 3241, 3081, 2957, 2922, 2851, 1743, 1662, 1560, 1476, 1257, 1171, 1041, 856, 775, 601 1 H NMR (DMSO-d6, 300 MHz) 9.44 (s, 1H); 8.91 (s, 1H); 8.23 (t, 1H, J = 5.63 Hz); 7.57 (s, 1H); 5.18 (s, 2H); 3.32 (d, 1H, J = 14.49 Hz); 3.20 (td, 2H, J1 = 6.44 Hz, J2 = 13.69 Hz); 2.88 (d, 1H, J = 14.49 Hz); 2.58­2.30 (m, 2H); 2.12­1.79 (m, 5H); 1.53 (t, 2H, J = 6.44Hz); 1.24 (s, 14H); 1.05 (s, 3H); 0,89-0,85 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.3; 164.2; 137.5; 134.4; 122.9; 67.2; 58.5; 51.6; 48.3; 47.9; 43.0; 42.6; 40.1; 31.9; 29.6; 29.3; 29.1; 27.0; 24.7; 22.7; 19.8; 19.7; 14.1. IR, /cm-1: 3447, 3240, 3080, 2957, 2922, 2851, 1743, 1662, 1559, 1476, 1257, 1170, 1041, 856, 776, 601. 1 H NMR (DMSO-d6, 300 MHz) 9.44 (s, 1H); 8.92 (s, 1H); 8.23 (t, 1H, J = 5.63 Hz); 7.57 (s, 1H); 5.19 (s, 2H); 3.31 (d, 1H, J = 14.47 Hz); 3.20 (td, 2H, J1 = 6.44 Hz, J2 = 13.34 Hz); 2.88 (d, 1H, J = 14.47 Hz); 2.57­2.31 (m, 2H); 2.12­1.79 (m, 5H); 1.53 (t, 2H, J = 6.44Hz); 1.24 (s, 18H); 1.05 (s, 3H); 0.89­0.85 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.3; 164.3; 137.5; 134.5; 123.0; 67.2; 58.5; 51.6; 48.3; 47.9; 43.0; 42.7; 40.1; 31.9; 29.6 (2C); 29.5; 29.3; 29.1; 27.0; 24.7; 22.7; 19.8; 19.7; 14.1. IR, /cm-1: 3447, 3241, 3080, 2957, 2922, 2851, 1743, 1662, 1559, 1476, 1257, 1170, 1041, 856, 776, 601. 1 H NMR (DMSO-d6, 300 MHz) 9.44 (s, 1H); 8.91 (s, 1H); 8.22 (t, 1H, J = 5.67 Hz); 7.57 (s, 1H); 5.18 (s, 2H); 3.32 (d, 1H, J = 14,49 Hz); 3.20 (td, 2H, J1 = 6.47 Hz, J2 = 13.62 Hz); 2.87 (d, 1H, J = 14.49 Hz); 2.57­2.31 (m, 2H); 2.12­1.79 (m, 5H); 1.53 (t, 2H, J = 6.44Hz); 1.24 (s, 22H); 1.05 (s, 3H); 0.89­0.85 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.3; 164.3; 137.5; 134.5; 123.0; 67.2; 58.5; 51.6; 48.3; 47.9; 43.0; 42.7; 40.1; 31.9; 29.6 (2C); 29.5; 29.4 (2C), 29.3; 29.1; 27.0; 24.7; 22.7; 19.8; 19.7; 14.1. H NMR (DMSO-d6, 300 MHz) 8.00 (s, 1H); 7.38 (s, 1H); 7.19 (s, 1H); 3.74 (d, 1H, J = 14.67 Hz); 3.19 (d, 1H, J = 14.67 Hz); 2.36-2.49 (m, 2H); 2.06-2.19 (m, 2H); 1.98 (d, 1H, J = 18.49 Hz); 1.73-1.82 (m, 1H); 1.50 (m, 1H); 1.14 (s, 3H); 0.89 (s, 3H). 13 C NMR (DMSO-d6, 75 MHz) 217.2; 138.3; 132.9; 119.2; 58.4; 48.2; 47.6; 43.1; 42.6; 25.7; 24.6; 19.8; 19.7. 1b 1c 2a 2b 2c ponent that consists mainly of lipopolysaccharides, which acts as a barrier and prevents antimicrobial agents and biocides from entering the cell (Pérez et al., 2009). The importance of structure for the initiation of biodegradation was studied by Boethling et al. (1989). They found a decrease of biodegradability under aerobic conditions in the following order: ester, amide, anhydride, hydroxyl and carboxyl. Considering the structure of prepared salts, derivatives 1a-1c with an ester functional group were only slightly effective as antimicrobial agents compared to amide analogues 2a-2c,, probably due to the faster biodegradation of ester derivatives. QASs exhibit strong antimicrobial activities and they are widely used as a disinfectants and antiseptics. The main target site of QASs is the cytoplasmic membrane comprised of a phospholipid bilayer. QASs are able to intercalate into the phospholipid bilayer, which is accompanied by membrane disorganization and structural and functional changes in the cell membrane, inducing leakage of intracellular components (Gilbert and Moore, 2005; Tischer et al., 2012; Wessels and Ingmer, 2013). In addition, QASs were found to inhibit ATP synthesis by neutralizing the proton motive force (PMF) (Denyer & Hugo, 1977). The PMF is initiated by a proton gradient across the cytoplasmic membrane and is involved in many respiratory and photosynthetic processes including ATP synthesis. QASs 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 also have intracellular targets and bind to DNA, which leads to the inhibition of DNA replication (Menzel et al., 2011; Zinchenko et al., 2004). On the other hand, for most of the QASs, no specific S. aureus E. coli C. albicans log 1/MIC 1a 1b 1c 2a 2b 2c BAB septonex Figure 2. Antimicrobial activity Log 1/MIC of camphorsulfonic acid derived imidazolium salts in series 1 and 2. target site has been recognized. However, it is not excluded CONCLUSIONS that there can exist some target specificities, as shown by In summary, we have designed and synthesized a new amphiMenzel (2011) and Zhang (2013), because the antimicrobial philic imidazolium salts that could be classified as potentially activity of QASs fluctuates significantly against various types "soft" antimicrobials. Salts 2a-2c with amide functional group of microorganisms and explanation simply by the cationic showed better antimicrobial and antifungal activity compared charge and hydrophobic tail cannot be used. The antimicro- to their ester analogues 1a-1c. The maximum antimicrobial bial activity of surfactants generally depends on the alkyl activity was observed for compounds with 12 carbon atoms chain length, although this correlation is not linear. In the in alkyl chain. Increasing the number of carbon atoms in alkyl series of prepared imidazolium salts 1a-1c and 2a-2c, maxi- chain decreased the biological activity of studied salts against mum antimicrobial activity was observed for compounds all microorganisms tested. The best antimicrobial activity, with 12 carbon atoms in alkyl chain 1a and 2a. Therefore, it shows 3-(2-decylamino-2-oxoethyl)-1-[(7,7-dimethyl-2-oxobican be inferred that increasing the number of carbon atoms cyclo[2.2.1]heptan-1-yl)methylsulfonyl]imidazolium bromide in alkyl chain decreased the biological activity of the studied 2a followed by 3-(2-dodecylamino-2-oxoethyl)-1-[(7,7-dimesalts against all microorganisms tested. thyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfonyl]imidazoIt is noteworthy that among the salts examined in this study, lium bromide 2b. Their activity was higher than clinically used the most active were 2a and 2b. Both compounds inhibited BAB. Nevertheless, they were less effective than the other the growth of microorganisms at the concentrations lower clinically used standard carbethopendecinium bromide. than BAB. On the other hand, none of the prepared salts was more effective than carbethopendecinium bromide. HowACKNOWLEDGEMENT ever, the medical use of QAS in some fields is limited by their high toxicity and low biodegradability. The "soft" cationic Financial support of this work by European Union in the amphiphilic compounds exhibit higher biodegradability project "Centrum excelentnosti bezpecnostného výskumu" and less toxicity that makes them more beneficial in medi- with code number 26240120034 is gratefully acknow cal applications despite slightly decreased biological activity. ledged by the authors. Table 3. MIC values (µmol l-1) of camphorsulfonic acid derived imidazolium salts of the compounds in series 1 and 2. Compound 1a 1b 1c 2a 2b 2c BAB Septonex S. aureus ATCC 6538 1391.1 5406.9 5560 5.4 10.4 80.4 26 4.7 E. coli CNCTC 377/79 2781.5 2703.5 5560 144.1 208 321.6 260 47.3 C. albicans CCM 8186 1391.1 1351.1 2780 5.4 10.4 40.2 26 1.9 46 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

Synthesis and antimicrobial properties of camphorsulfonic acid derived imidazolium salts

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Abstract

Keywords Kúcové slová: imidazolium salts, antimicrobial activity, camphorsulfonic acid imidazóliové soli, antimikróbna aktivita, kyselina gáforsulfónová INTRODUCTION The strong bactericidal activity of quaternary ammonium salts (QAS) with long alkyl chains have been known since 1915 (Jacobs & Heidelberger, 1915) and studied further on a broad range of microorganisms such as Gram positive (G+) and Gram negative (G-) bacteria and fungi (Lukác et al., 2010; Miklás et al., 2012, 2014), certain viruses (Wong et al., 2002), herbicidal salts (Cojocaru et al., 2013) and even anticancer agents (Kaushik et al., 2012). 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 (Devínsky et al., 1987; Mlynarcík et al., 1981). The widespread importance of surfactants in practical applications (Brak & Jacobsen, 2013; Cortesi et al., 2012; Gilbert & Moore, 2005; Zhi et al., 2012) and difficulties to recover or reuse them due to their water solubil* miklas@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae ity causes their accumulation in environment, thus affecting aquatic ecosystem. Most QASs are not easily biodegraded; therefore, the aquatic microorganisms remain in contact with them for a longer period of time, which increases their toxicity. It was found (Kümmerer et al., 1997) that LC50 of benzalkonium chloride to fish was between 0.5 and 5.0 mg l-1 and to daphnids even from 0.1 to 1.0 mg l-1. In addition, the presence of QASs may decrease the biodegradation efficiency of linear alkylbenzene sulfonates. The toxicity of QASs depends on both polar and hydrophobic parts of the molecule. Generally, the double-tail surfactants are less toxic than the single-tail counterparts (Pinnaduwage et al., 1989). The length of the hydrophobic alkyl chain in QASs plays also an important role for toxicity. While for aliphatic single-tail cationic surfactants, the toxicity increases with the increasing alkyl chain length; (Rasia et al., 2007) for Acetone was distilled from potassium carbonate. Bromoacetic double-tail cationic surfactant, the toxicity decreases as the acid esters (Michniak et al., 1996) and amides (Hoque et al., alkyl chain length increases (Spelios & Savva, 2008). Concern- 2012) were synthesized by modification of published proing the polar part of the QAS`s molecule, the positive charge cedures. 1H and 13C NMR spectra were measured on a Varian can be located on a quaternary ammonium group or delo- Gemini 300 spectrometer at 300 MHz and 75 MHz, respectively. calized in heterocyclic ring or in a guanidine group. Delocali- Chemical shifts have been reported in ppm relative to an interzation of the positive charge mostly helps to decrease the nal reference (TMS). IR spectra (in KBr pellets) were recorded toxicity of such surfactants (Coleman et al., 2012). In order to on FTIR Impact 400D Nicolet instrument. Polarimetric measavoid systemic toxicity and environmental persistency of QAS urements were obtained using a Jasco P-1010 polarimeter at antimicrobials, the "soft" drug approach developed by Bodor 589 nm. Elemental analyses were carried out on a Carlo Erba et al. (1980) was applied to this problem. The structural "soft" 1108A instrument. All melting points reported were uncorrectanalogues, compared to their "hard" counterparts, have a spe- ed and measured on Kofler hot stage. cific easily degradable functional group built into their structures to provide their one-step detoxification. Various "soft" 2.2 Microbiology surfactants possessing a cleavable moiety, such as an ester The antimicrobial activity was tested against Gram-negative and/or amide bond, have been synthesized and their antimi- bacteria Escherichia coli CNCTC 377/79, Gram-positive baccrobial activity and biodegradation was tested (Colomer et al., teria Staphylococcus aureus ATCC 6538 and fungi Candida al2012; Devínsky et al., 1991; Fan et al., 2013). The well-known bicans CCM 8186. Solutions of the compounds studied were antibacterial effect of essential oils containing bicyclic cam- prepared in DMSO (5%, w/v). A suspension of the standard phor or borneol (avar et al., 2012; Ruiz-Navajas et al., 2012) microorganism, prepared from 24-h cultures of bacteria in resulted in the idea to design and synthesize potentially "soft" blood agar and from 24-h cultures in the Sabouraud agar for QASs bearing hydrophobic chiral camphor derived moieties, fungi had a concentration of 5x107 cfu ml-1 of bacteria and hoping that incorporation of two important antimicrobial ac- 5x105 cfu ml-1 of Candida. Concentration of microorganisms tive structures in one compound will improve their bioactivity. were determined spectrophotometrically at 540 nm and adIn this study, we have prepared a series of six new optically justed to absorbance A = 0.35. The microorganism suspenactive quaternary imidazolium salts, which could be specified sion (5 l) was added to solutions containing the compound as potentially "soft" disinfectants due to the ester or amide under examination (100 l) and to double-concentrated pepbonds in the structure (Fig. 1). Their antimicrobial activity was tone broth medium (8%) for bacteria or Sabouraud medium tested against Gram-negative Escherichia coli, Gram-positive (12%) for Candida (100 l).The double-concentrated medium human pathogenic bacteria Staphylococcus aureus and hu- was used due to addition of the same volume of solution man fungal pathogen Candida albicans. containing the compound tested, which resulted in the desired standard concentration of medium for experiments. The stock solution of tested compounds was serially diluted by 2. EXPERIMENTAL half. The cultures were carried out using 96-well microliter 2.1 Materials and methods plates. The microorganisms were incubated for 24 h at 37°C All compounds used ((1S)-(+)-camphor-10-sulfonic acid (CSA), and then from each well, 5 l of suspension were cultured on thionylchloride, triethylamine (TEA), diethyl ether, acetone, blood agar (bacteria) or on Sabouraud agar (fungi). After 24 h dichloromethane (DCM), ethyl acetate, petroleum ether (40­ at 37°C, the lowest concentration of QAS, which prevented 65°C), DMSO, bromoacetyl bromide, fatty alcohols and amines) colony formation, was determined as minimal inhibitory conare commercially available. DCM was pre-dried over CaCl2 and centration (MIC). Benzalkonium bromide (BAB, Ajatin®) and then distilled from CaH2 under a nitrogen atmosphere. Diethyl carbethopendecinium bromide (Septonex®) were used as the ether was pre-dried from KOH and then distilled from sodium. standards. H N SOCl2 DCM, TEA SO2Cl R-Br CH3CN O2 S O N 100°C O2 S O N SO3H 1a 1b 1c 2a 2b 2c N+ R R = -CH2COO-decyl R = -CH2COO-dodecyl R = -CH2COO-tetradecyl R =-CH2CONH-decyl R = -CH2CONH-dodecyl R = -CH2CONH-tetradecyl Figure 1. Preparation of camphorsulfonic acid derived imidazolium salts of the compounds in series 1 and 2. 2.3 Synthesis Enantiopure camphor sulfonylchloride 4 was prepared according to the known procedure (Gayet et al., 2004). 1-((1H-imidazol-1-ylsulfonyl)methyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one (3) To a solution of imidazole (7.1 g, 0.104 mol) and TEA (11.1 ml, 0.08 mol) in anhydrous DCM (50 ml) was added drop wise a solution of 4 (20.12 g, 0.08 mol) in anhydrous DCM (80 ml) at 0°C over 30 min. After the addition was complete, the reaction mixture was heated to reflux for 1.5 h. and then stirred overnight at ambient temperature. A precipitate was filtered off and filtrate was extracted with 20% (w/v) aqueous solution of Na2CO3 (2 x 50 ml) and brine (50 ml). The organic layer was dried over anhydrous Na2SO4 and evaporated to yield a yellowish solid. The crude product was recrystallized from acetone/petroleum ether (3:1, v/v) mixture to yield 19.1 g (84%) of white crystals. General procedure for the synthesis of quaternary salts 1 and 2 The sulfonamide 3 (12 mmol) was mixed with 1.2 equivalents of the appropriate alkylating bromoderivative in CH3CN (20 ml). Reaction mixture was stirred at ambient temperature for 2 h., then refluxed for 24 h. and allowed to cool. The reaction mixture was placed in the freezer for 2 days and the resulting crystals were filtered off, washed twice with 25 ml of anhydrous diethyl ether and the crude product was recrystallized repeatedly from anhydrous acetone. Characterisation and spectral data of the prepared salts are sumarized in Tables 1 and 2. 3. RESULTS AND DISCUSSION Enantiopure QASs of the compounds in series 1 and 2 were synthesized as illustrated in Fig. 1. starting from (1S)-camphor10-sulfonic acid. Although sulfonylchloride 4 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 4 by the published procedure (Gayet et al., 2004). Thus (1S)-camphor-10-sulfonic acid was reacted with thionylchloride providing compound 4 in 86% yield after crystallization from petroleum ether. The preparation of camphor sulfonamide 3 was carried out by dropwise addition of 4 in anhydrous DCM into the solution of imidazole in DCM in the presence of TEA as a base. Two series of imidazolium salts 1 and 2 were formed after quaternization of 3 by n-alkyl esters or N-alkyl amides of bromoacetic acid (alkyl = decyl, dodecyl, tetradecyl) in acetonitrile. All imidazolium salts were obtained as colorless crystals after several crystallizations from anhydrous acetone in yields ranging from 24 to 46%. They were identified and characterized thoroughly from spectral and analytical data. The antimicrobial activities of imidazolium salts, were determined as a MIC, [mol 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 3. The MIC values were determined as lowest concentration of the imidazolium salt that completely prevented visible colony formation. All the compounds were dissolved in DMSO for biological evaluation. 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 inhibitory activity. Clinically used benzalkonium bromide (BAB, Ajatin®) and carbethopendecinium bromide (Septonex®) were used as standards. According to the results, it can be observed that all of the synthesized imidazolium salts exhibit growth inhibition effect against all three types of microbes, with higher efficiency against S. aureus and C. albicans (Fig. 2). Gram-negative E. coli was found to be most resistant to the prepared salts, presumably due to the cell membrane composition. Gram-negative bacteria contain an outer membrane with an external com- Table 1. Characterisation of camphorsulfonic acid derived imidazolium salts of the compounds in series 1 and 2 Compound Formula / []D21 (conc., solvent) C25H41BrN2O5S/ - 0.86 (0.1 g/100 ml, CHCl3) C27H45BrN2O5S/ - 0.44 (0.1 g/100 ml, CHCl3) C29H49BrN2O5S/ - 0.34 (0.1 g/100 ml, CHCl3) C25H42BrN3O4S/ - 9.34 (0.1 g/100 ml, CHCl3) C27H46BrN3O4S/ - 6.12 (0.1 g/100 ml, CHCl3) C29H50BrN3O4S/ - 5.47 (0.1 g/100 ml, CHCl3) C13H18N2O3S/ - 15.5 (0.1 g/100 ml, CHCl3) wi(calc.)/% wi(found)/% C 53.47 53.29 55.00 54.91 56.39 56.22 53.56 53.51 55.09 54.94 56.48 56.39 55.30 55.12 H 7.36 7.43 7.69 7.84 8.00 8.06 7.55 7.39 7.88 7.83 8.17 8.24 6.43 6.51 N 4.99 5.11 4.75 4.88 4.54 4.68 7.50 7.67 7.14 7.21 6.81 6.74 9.92 9.78 S 5.71 5.58 5.44 5.20 5.19 5.02 5.72 5.91 5.45 5.69 5.20 5.44 11.36 11.49 Yield % 46 38 41 24 33 38 84 M.p. °C 153­155 141­143 137­138 134­135 123­124 113­114 173­175 1a 1b 1c 2a 2b 2c 3 Table 2. Spectroscopic data of camphorsulfonic acid derived imidazolium salts of the compounds in series 1 and 2 Compound 1a Spectral data IR, /cm-1: 3057, 2957, 2922, 2852, 1751,1563, 1467, 1234, 1183,1042, 969, 759, 601 1H NMR (DMSO-d6, 300 MHz) 10.14 (s, 1H); 8.62 (s, 1H); 7.63 (s, 1H); 5.37 (s, 2H); 4.18 (t, 2H, J =7.2 Hz); 3.48 (d, 1H, J = 14.83 Hz); 2.88 (d, 1H, J = 14.83 Hz); 2.39­2.28 (m, 2H); 2.11­1.87 (m, 5H); 1.66 (t, 2H, J = 6.87Hz); 1.26 (s, 14H); 1.05 (s, 3H); 0.9­0.83 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.1; 165.8; 139.3; 133.5; 123.2; 67.1; 58.4; 50.3; 48.1; 47.6; 43,0; 42.5; 31.8; 29.5; 29.4; 29.3; 29.2; 28.3; 27.0; 25.7; 24.5; 22.6; 19.8; 19.7; 14.1. IR, /cm-1: 3111, 2957, 2921, 2851, 1749, 1583, 1468, 1399, 1374, 1236, 1182, 1041, 968, 866, 763, 601 1 H NMR (DMSO-d6, 300 MHz) 10.13 (s, 1H); 8.61 (s, 1H); 7.63 (s, 1H); 5.36 (s, 2H); 4.18 (t, 2H, J =7,2 Hz); 3.48 (d, 1H, J = 14.84 Hz); 2.88 (d, 1H, J = 14.84 Hz); 2.39­2.28 (m, 2H); 2.11­1.87 (m, 5H); 1.66 (t, 2H, J = 6.87Hz); 1.26 (s, 18H); 1.05 (s, 3H); 0.9­0.83 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.1; 165.9; 139.3; 133.5; 123.3; 67.1; 58.4; 50.3; 48.1; 47.6; 43,0; 42.6; 31.9; 29.7; 29.6; 29.5; 29.4; 29.3; 29.2; 28.3; 27.0; 25.7; 24.6; 22.7; 19.8; 19.7; 14.1. IR, /cm-1: 3111, 2956, 2920, 2851, 1748, 1584, 1468, 1396, 1374, 1234, 1182, 1042, 966, 788, 601 1 H NMR (DMSO-d6, 300 MHz) 10.13 (s, 1H); 8.61 (s, 1H); 7.63 (s, 1H); 5.36 (s, 2H); 4.18 (t, 2H, J =7,2 Hz); 3.48 (d, 1H, J = 14.84 Hz); 2.88 (d, 1H, J = 14.84 Hz); 2.39­2.28 (m, 2H); 2.11­1.87 (m, 5H); 1.66 (t, 2H, J = 6.87Hz); 1.26 (s, 22H); 1.05 (s, 3H); 0.9­0.83 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.1; 166.0; 139.5; 133.8; 123.3; 67.1; 58.5; 50.3; 48.1; 47.7; 43.0; 42.6; 31.9; 29.7(2C); 29.6; 29.5; 29.4; 29.2; 28.4; 27.0; 25.7; 24.6; 22.7; 19.8; 19.7; 14.1. IR, /cm-1: 3447, 3241, 3081, 2957, 2922, 2851, 1743, 1662, 1560, 1476, 1257, 1171, 1041, 856, 775, 601 1 H NMR (DMSO-d6, 300 MHz) 9.44 (s, 1H); 8.91 (s, 1H); 8.23 (t, 1H, J = 5.63 Hz); 7.57 (s, 1H); 5.18 (s, 2H); 3.32 (d, 1H, J = 14.49 Hz); 3.20 (td, 2H, J1 = 6.44 Hz, J2 = 13.69 Hz); 2.88 (d, 1H, J = 14.49 Hz); 2.58­2.30 (m, 2H); 2.12­1.79 (m, 5H); 1.53 (t, 2H, J = 6.44Hz); 1.24 (s, 14H); 1.05 (s, 3H); 0,89-0,85 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.3; 164.2; 137.5; 134.4; 122.9; 67.2; 58.5; 51.6; 48.3; 47.9; 43.0; 42.6; 40.1; 31.9; 29.6; 29.3; 29.1; 27.0; 24.7; 22.7; 19.8; 19.7; 14.1. IR, /cm-1: 3447, 3240, 3080, 2957, 2922, 2851, 1743, 1662, 1559, 1476, 1257, 1170, 1041, 856, 776, 601. 1 H NMR (DMSO-d6, 300 MHz) 9.44 (s, 1H); 8.92 (s, 1H); 8.23 (t, 1H, J = 5.63 Hz); 7.57 (s, 1H); 5.19 (s, 2H); 3.31 (d, 1H, J = 14.47 Hz); 3.20 (td, 2H, J1 = 6.44 Hz, J2 = 13.34 Hz); 2.88 (d, 1H, J = 14.47 Hz); 2.57­2.31 (m, 2H); 2.12­1.79 (m, 5H); 1.53 (t, 2H, J = 6.44Hz); 1.24 (s, 18H); 1.05 (s, 3H); 0.89­0.85 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.3; 164.3; 137.5; 134.5; 123.0; 67.2; 58.5; 51.6; 48.3; 47.9; 43.0; 42.7; 40.1; 31.9; 29.6 (2C); 29.5; 29.3; 29.1; 27.0; 24.7; 22.7; 19.8; 19.7; 14.1. IR, /cm-1: 3447, 3241, 3080, 2957, 2922, 2851, 1743, 1662, 1559, 1476, 1257, 1170, 1041, 856, 776, 601. 1 H NMR (DMSO-d6, 300 MHz) 9.44 (s, 1H); 8.91 (s, 1H); 8.22 (t, 1H, J = 5.67 Hz); 7.57 (s, 1H); 5.18 (s, 2H); 3.32 (d, 1H, J = 14,49 Hz); 3.20 (td, 2H, J1 = 6.47 Hz, J2 = 13.62 Hz); 2.87 (d, 1H, J = 14.49 Hz); 2.57­2.31 (m, 2H); 2.12­1.79 (m, 5H); 1.53 (t, 2H, J = 6.44Hz); 1.24 (s, 22H); 1.05 (s, 3H); 0.89­0.85 (m, 6H). 13 C NMR (DMSO-d6, 75 MHz) 217.3; 164.3; 137.5; 134.5; 123.0; 67.2; 58.5; 51.6; 48.3; 47.9; 43.0; 42.7; 40.1; 31.9; 29.6 (2C); 29.5; 29.4 (2C), 29.3; 29.1; 27.0; 24.7; 22.7; 19.8; 19.7; 14.1. H NMR (DMSO-d6, 300 MHz) 8.00 (s, 1H); 7.38 (s, 1H); 7.19 (s, 1H); 3.74 (d, 1H, J = 14.67 Hz); 3.19 (d, 1H, J = 14.67 Hz); 2.36-2.49 (m, 2H); 2.06-2.19 (m, 2H); 1.98 (d, 1H, J = 18.49 Hz); 1.73-1.82 (m, 1H); 1.50 (m, 1H); 1.14 (s, 3H); 0.89 (s, 3H). 13 C NMR (DMSO-d6, 75 MHz) 217.2; 138.3; 132.9; 119.2; 58.4; 48.2; 47.6; 43.1; 42.6; 25.7; 24.6; 19.8; 19.7. 1b 1c 2a 2b 2c ponent that consists mainly of lipopolysaccharides, which acts as a barrier and prevents antimicrobial agents and biocides from entering the cell (Pérez et al., 2009). The importance of structure for the initiation of biodegradation was studied by Boethling et al. (1989). They found a decrease of biodegradability under aerobic conditions in the following order: ester, amide, anhydride, hydroxyl and carboxyl. Considering the structure of prepared salts, derivatives 1a-1c with an ester functional group were only slightly effective as antimicrobial agents compared to amide analogues 2a-2c,, probably due to the faster biodegradation of ester derivatives. QASs exhibit strong antimicrobial activities and they are widely used as a disinfectants and antiseptics. The main target site of QASs is the cytoplasmic membrane comprised of a phospholipid bilayer. QASs are able to intercalate into the phospholipid bilayer, which is accompanied by membrane disorganization and structural and functional changes in the cell membrane, inducing leakage of intracellular components (Gilbert and Moore, 2005; Tischer et al., 2012; Wessels and Ingmer, 2013). In addition, QASs were found to inhibit ATP synthesis by neutralizing the proton motive force (PMF) (Denyer & Hugo, 1977). The PMF is initiated by a proton gradient across the cytoplasmic membrane and is involved in many respiratory and photosynthetic processes including ATP synthesis. QASs 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 also have intracellular targets and bind to DNA, which leads to the inhibition of DNA replication (Menzel et al., 2011; Zinchenko et al., 2004). On the other hand, for most of the QASs, no specific S. aureus E. coli C. albicans log 1/MIC 1a 1b 1c 2a 2b 2c BAB septonex Figure 2. Antimicrobial activity Log 1/MIC of camphorsulfonic acid derived imidazolium salts in series 1 and 2. target site has been recognized. However, it is not excluded CONCLUSIONS that there can exist some target specificities, as shown by In summary, we have designed and synthesized a new amphiMenzel (2011) and Zhang (2013), because the antimicrobial philic imidazolium salts that could be classified as potentially activity of QASs fluctuates significantly against various types "soft" antimicrobials. Salts 2a-2c with amide functional group of microorganisms and explanation simply by the cationic showed better antimicrobial and antifungal activity compared charge and hydrophobic tail cannot be used. The antimicro- to their ester analogues 1a-1c. The maximum antimicrobial bial activity of surfactants generally depends on the alkyl activity was observed for compounds with 12 carbon atoms chain length, although this correlation is not linear. In the in alkyl chain. Increasing the number of carbon atoms in alkyl series of prepared imidazolium salts 1a-1c and 2a-2c, maxi- chain decreased the biological activity of studied salts against mum antimicrobial activity was observed for compounds all microorganisms tested. The best antimicrobial activity, with 12 carbon atoms in alkyl chain 1a and 2a. Therefore, it shows 3-(2-decylamino-2-oxoethyl)-1-[(7,7-dimethyl-2-oxobican be inferred that increasing the number of carbon atoms cyclo[2.2.1]heptan-1-yl)methylsulfonyl]imidazolium bromide in alkyl chain decreased the biological activity of the studied 2a followed by 3-(2-dodecylamino-2-oxoethyl)-1-[(7,7-dimesalts against all microorganisms tested. thyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfonyl]imidazoIt is noteworthy that among the salts examined in this study, lium bromide 2b. Their activity was higher than clinically used the most active were 2a and 2b. Both compounds inhibited BAB. Nevertheless, they were less effective than the other the growth of microorganisms at the concentrations lower clinically used standard carbethopendecinium bromide. than BAB. On the other hand, none of the prepared salts was more effective than carbethopendecinium bromide. HowACKNOWLEDGEMENT ever, the medical use of QAS in some fields is limited by their high toxicity and low biodegradability. The "soft" cationic Financial support of this work by European Union in the amphiphilic compounds exhibit higher biodegradability project "Centrum excelentnosti bezpecnostného výskumu" and less toxicity that makes them more beneficial in medi- with code number 26240120034 is gratefully acknow cal applications despite slightly decreased biological activity. ledged by the authors. Table 3. MIC values (µmol l-1) of camphorsulfonic acid derived imidazolium salts of the compounds in series 1 and 2. Compound 1a 1b 1c 2a 2b 2c BAB Septonex S. aureus ATCC 6538 1391.1 5406.9 5560 5.4 10.4 80.4 26 4.7 E. coli CNCTC 377/79 2781.5 2703.5 5560 144.1 208 321.6 260 47.3 C. albicans CCM 8186 1391.1 1351.1 2780 5.4 10.4 40.2 26 1.9 46

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Dec 30, 2014

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