Quaternary salts derived from 3-substituted quinuclidine as potential antioxidative and antimicrobial agents

Article abstract of DOI:10.1515/chem-2017-0031

Two series of novel ammonium salts containing the quinuclidine moiety were prepared in order to evaluate their antioxidative, antibacterial and antifungal potential. The synthesized homologues of 3-hydroxy (QOH) and 3-chloroquinuclidine (QCl) with the different N-benzyl substituents at the para-position (bromo, chloro or nitro group) were obtained in very good yields and characterized by IR and NMR spectroscopies and elemental analysis. All compounds were tested for antioxidative activity using the oxygen radical absorbance capacity (ORAC) assay and among tested samples, N-p-nitrobenzyl-3-hydroxyquinuclidinium bromide (QOH-4) exhibited the highest antioxidative potential (293.80 nmol (TE) mL^-1), which was further investigated by the DNA nicking assay. The biological activity of selected compounds was evaluated by measuring the zone of inhibition and by determining the minimal inhibitory concentration (MIC) against three Gram-positive bacteria (B. cereus, E. faecalis and S. aureus), three Gram-negative bacteria (E. coli, P. aeruginosa and C. sakazakii) and three fungi species (C. albicans, A. Niger and P. notatum). The bioactivity assay showed that some newly synthetized quaternary quinuclidinium compounds display a comparable or even better antibacterial and antifungal activity than the reference drugs such as gentamicin (GEN), cefotaxime (CTX) and amphotericin B (AMPHB). Among the tested compounds, N-p-chlorobenzyl-3-hydroxyquinuclidinium bromide (QOH-3) exhibited a considerable antibacterial efficiency against P. aeruginosa (MIC=0.39 μg mL^-1) and QOH-4 displayed a potent antifungal activity against C. albicans (MIC=1.56 μg mL^-1).

Full text of DOI:10.1515/chem-2017-0031

Open Chem., 2017; 15: 320–331
Research Article
Open Access
Renata Odžak*, Matilda Šprung, Barbara Soldo, Mirjana Skočibušić, Martina Gudelj, Anita
Muić, Ines Primožič
Quaternary salts derived from 3-substituted
quinuclidine as potential antioxidative and
antimicrobial agents
https://doi.org/10.1515/chem-2017-0031
received September 4, 2017; accepted October 21, 2017.
compounds, N-p-chlorobenzyl-3-hydroxyquinuclidinium
bromide (QOH-3) exhibited a considerable antibacterial
Abstract: Two series of novel ammonium salts containing efficiency against P. aeruginosa (MIC=0.39 µg mL-1) and
the quinuclidine moiety were prepared in order to QOH-4 displayed a potent antifungal activity against C.
evaluate their antioxidative, antibacterial and antifungal albicans (MIC=1.56 µg mL-1).
potential. The synthesized homologues of 3-hydroxy
(QOH) and 3-chloroquinuclidine (QCl) with the different Keywords: quaternary ammonium salts, ORAC assay,
N-benzyl substituents at the para-position (bromo, chloro DNA damage protection activity, antioxidative and
or nitro group) were obtained in very good yields and antimicrobial activity
characterizedbyIRandNMRspectroscopiesandelemental
analysis. All compounds were tested for antioxidative
activity using the oxygen radical absorbance capacity 1 Introduction
(ORAC) assay and among tested samples, N-p-nitrobenzyl-
3-hydroxyquinuclidinium bromide (QOH-4) exhibited Quinuclidine (1-azabicyclo[2.2.2]octane) is found to be
the highest antioxidative potential (293.80 nmol (TE) a part of the structure of numerous naturally occurring
mL-1), which was further investigated by the DNA nicking bioactive compounds, such as cinchona, indole
assay. The biological activity of selected compounds was alkaloids and synthetic drugs [1-3]. Many clinically
evaluated by measuring the zone of inhibition and by useful chemotherapeutic agents contain quinuclidine
determining the minimal inhibitory concentration (MIC) nucleus as an essential part of their structure and several
against three Gram-positive bacteria (B. cereus, E. faecalis scientific papers regarding its synthesis and/or biological
and S. aureus), three Gram-negative bacteria (E. coli, P. activity have been published [4]. Recently some synthetic
aeruginosa and C. sakazakii) and three fungi species quinuclidines have been identified as a specific muscarinic
(C. albicans, A. niger and P. notatum). The bioactivity agonists with potential in Alzheimer’s dementia therapy
assay showed that some newly synthetized quaternary [1-5] and some 3-substitued quinuclidine derivatives have
quinuclidinium compounds display a comparable or been shown to be potential antidotes against poisoning
even better antibacterial and antifungal activity than the by organophosphorus compounds such as pesticides,
reference drugs such as gentamicin (GEN), cefotaxime insecticides and chemical warfare agents [1-3], [5]. Today,
(CTX) and amphotericin B (AMPHB). Among the tested few esters of quinuclidine-3-ol are even commercially
available as therapeutic agents [6], such as zacopride and
RG12915,whichareclassical5-hydroxytryptamine3(5-HT3)
receptor antagonists [7-10]. In addition, an antimicrobial
*Corresponding author: Renata Odžak: Department of Chemistry,
activity of a quinuclidine-based FtsZ inhibitor and its
synergistic potential with β-lactam antibiotics has been
reported. FtsZ is an essential GTP-ase in bacterial cell
division which is highly conserved among Gram-positive
and Gram-negative bacteria. Therefore, it represents an
interesting molecular target that could be manipulated in
order to develop new antibacterial agents [11-12].
Faculty of Science, University of Split, R. Boškovića 33, 21 000 Split,
E-mail: rodzak@pmfst.hr
Matilda Šprung, Barbara Soldo, Martina Gudelj, Anita Muić:
Department of Chemistry, Faculty of Science, University of Split, R.
Boškovića 33, 21 000 Split
Mirjana Skočibušić: Department of Biology, Faculty of Science,
University of Split, R. Boškovića 33, 21 000 Split
Ines Primožič: Department of Chemistry, Faculty of Science,
University of Zagreb, Horvatovac 102a, 10 000 Zagreb
Open Access. © 2017 Renata Odžak et al., published by De Gruyter Open.
Attribution-NonCommercial-NoDerivatives 4.0 License.
This work is licensed under the Creative Commons
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It is well known that during the process of cell have shown the best antioxidative and DNA protection
respiration, reactive oxygen species (ROS) are generated properties with a promising potential in the development
as byproducts of normal metabolism. The accumulation of quinuclidine QAS with new applications in medicine,
of ROS results in the damage of essential cellular pharmacy and the food industry.
compartments and biomolecules (proteins, DNA, fatty
acids) which can ultimately lead to the development of
serious pathological conditions. Cells have evolutionarily
2 Experimental procedure
developed several ROS defending mechanisms such as
enzymes superoxide dismutase, glutathione reductase,
and catalase. However, the ability to effectively neutralize 2.1 Synthesis
ROSbyacquiredmechanismsgraduallydecreaseswithcell
aging; therefore an external consumption of antioxidants Quaternization reagents were obtained from Sigma-
able to scavenge ROS is principally recommended [13]. The Aldrich. Dry solvents were used throughout the synthesis.
most popular natural source of antioxidants are plants and Reactions were monitored by thin layer chromatography
their extracts, but recently some synthetic forms have also using DC-Alufolien Aluminiumoxide 60 F254 plates (Merck)
gained much attention such as stobadine and derivatives with 9:1 chloroform/methanol as the eluent. The detection
of dihydropyridine [14].
of spots was achieved by UV light and by the reversible
A variety of quaternary ammonium salts (-N+RR’R’’, absorption of iodine. Melting points were determined
QAS) containing different heterocyclic moieties and in open capillaries using a Büchi B-540 apparatus and
synthesized via the Menschutin reaction have been are uncorrected. Elemental analyses were performed
reported for an increased pharmacological potential with a PerkinElmer PE 2400 Series II CHNS/O Analyser.
[15-19]. As such, chitin and chitosan have lately become FTIR spectra were recorded on a PerkinElmer FTIR 1725
extensively popular because of their broad application X spectrometer. All samples were prepared by mixing
possibilities in medicine and several other industries. FTIR-grade KBr (Sigma-Aldrich) with 1% (w/w) salt and
Chitosan is a nitrogeneous polysaccharide derived grinding to a fine powder. Spectra were recorded over
from chitin by a process of N-deacetylation. In the work the 400-4000 cm-1 range without baseline corrections.
reported by Liu et al. the antioxidative potential of 1H and 13C 1D NMR spectra were recorded in DMSO-d6
different chitosan derivatives was explored, among which solutions with a Bruker Avance III HD 400 MHz/54 mm
QAS have shown the highest activity [20]. This increased Ascend spectrometer (400 MHz) at room temperature.
activity was attributed to the positive charge density of Chemical shifts are reported as δ values in ppm using
the nitrogen atom in QAS which was later explored by the TMS as an internal standard. Abbreviations for data
introduction of the different substituting groups, namely quoted are: s, singlet; d, doublet; dd, doublet of doublets
highly electronegative –CBr3 and –CCl3 [21]. These studies and m, multiplet. Coupling constants (J) are given in Hz.
have shown that QAS have strong free radical scavenging Atomic numbering of quinuclidine heterocyclic ring is in
1
13
activity and that this property could be modulated by the accordance with the IUPAC recommendations. H and C
introduction of the different electronegative substituents NMR spectra are given in Supporting information. NMR
that would further strengthen the positive charge on the spectroscopic signals were assigned after reference to the
QAS nitrogen atom.
literature [24,25]
Given the persistent incidence of a bacterial multidrug
resistance and the antimicrobial potency of QAS that we
have previously shown with imidazole and quinuclidine 2.2 General procedure for synthesized
moieties[22-23],inthispaperwehavedesignedandexplored
new quinuclidine compounds, 3-hydroxyquinuclidine
compounds
and 3-chloroquinuclidine (QOH and QCl) quaternized by To the solution of 3-hydroxyquinuclidine (1.00 mmol)
benzylderivatives. Inordertoexploretheeffectofapositive in dry acetone, an equimolar amount of the appropriate
charge density of the nitrogen atom on antioxidative and quaternizationreagent(benzylbromide,para-chlorobenzyl
antimicrobial activities, three powerful electronegative bromide,para-bromobenzylbromideandpara-nitrobenzyl
groups -Br, -Cl and -NO2 were introduced into the para bromide, respectively) was added at room temperature.
position of N-benzyl-3-substituted quinuclidinium salts. The reaction mixture was kept in the dark for 5 days to
The compound with -NO2 group at para position on the obtain a solid product. The excess of the solvent was then
benzyl ring and -OH group on the quinuclidine ring removed under reduced pressure and the solid was washed
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several times with dry diethyl ether to give quaternary 7.78 - 7.89 (m, 2 H, H2’ and H6’) 8.31 - 8.40 (m, 2 H, H3’ and
compounds as white crystals. The QCl-1-4 crystals were H5’); 13C NMR (101 MHz, DMSO-d6) δ/ppm 17.81 (C5), 21.35
synthesized following the same procedure as for QOH-1-4 (C8), 27.13 (C4), 53.30 (C6), 54.61 (C7), 63.32 (C2), 63.74 (C3),
by using 3-chloroquinuclidine with prior removal of HCl. 64.92 (CH2-Bn), 124.27 (C3’ and C5’), 135.11 (C2’ and C6’),
All crystals were obtained in very good yields.
149.02 (C1’). Anal. calcd. for C14H19BrN2O3: C 48.99; H 5.58;
N-benzyl-3-hydroxyquinuclidinium bromide (QOH-1). Br 23.28; N 8.16; O 13.98. Found: C 49.01, H 5.58, N 8.15.
Yield: 89.5%; mp: 178.4-180.3°C; IR (KBr) v/cm-1: 3500-
N-benzyl-3-chloroquinuclidinium bromide (QCl-1).
3100, 3002, 2940, 1466, 1120, 1105, 1079, 1040, 1011, 951, Yield: 80.0%; mp: 197.9-199.3°C; IR (KBr) v/cm-1: 3002,
1
941, 595. H NMR (400 MHz, DMSO-d6) δ/ppm 1.67 – 2.06 2955-2851, 1530, 1460, 1322, 952, 844; 700, 620. 1H NMR
(m, 5 H, H4, H5, H8), 3.03 – 3.08 (m, 1 H, H2b), 3.26 - 3.50 (400 MHz, DMSO-d6) δ/ppm 1.84 – 2.35 (m, 5 H, H4, H5
(m, 4H, H6 and H7), 3.62 - 3.70 (m, 1 H, 2a), 4.06 (m, 1 H, and H8), 3.27 - 3.67 (m, 5 H, H2a, H6 and H7), 3.97 - 4.08
H3), 4.44 - 4.54 (m, 2 H, CH2-Bn), 5.54 (d, J=3.67 Hz, 1 H, (m, 1 H, H2b), 4.55 (s, 2 H, CH2-Bn), 4.69 - 4.77 (m, 1 H,
OH), 7.49 - 7.56 (m, 5 H, Ar); 13C NMR (101 MHz, DMSO-d6) δ/ H3), 7.47 - 7.59 (m, 5 H, Ar);13C NMR (101 MHz, DMSO-d6) δ/
ppm 17.80 (C5) 21.37 (C8), 27.24 (C4), 53.23 (C6), 54.24 (C7), ppm 18.71 (C5), 22.77 (C8), 28.35 (C4), 52.45 (C6), 54.09 (C3),
63.02 (C2),63.75 (C3), 66.44 (CH2-Bn), 128.09 (C1’), 129.41 54.23 (C7), 62.03 (C2), 66.34 (CH2-Bn), 127.72 (C1’), 129.51
(C3’ and C5’), 130.62 (C4’), 133.53 (C2’ and C6’). Anal. calcd. (C5’ and C3’), 130.78 (C4’), 133.57 (C2’ and C6’). Anal. calcd.
for C14H20BrNO: C 56.38; H 6.76; Br 26.79; N 4.70; O 5.36. for C14H19BrClN: C 53.10; H 6.05; Br 25.23; Cl 11.20; N 4.42.
Found: C 56.36, H 6.76, N 4.71.
Found: C 53.11, H 6.04, N 4.43.
N-p-bromobenzyl-3-hydroxyquinuclidinium bromide
N-p-bromobenzyl-3-chloroquinuclidinium bromide
(QOH-2). Yield: 98.0%; mp: 243.6-245.0°C; IR (KBr) v/cm-1: (QCl-2). Yield: 88.6%; mp: 219.7-221.3°C; IR (KBr) v/cm-1:
3526-3158, 3012, 2942, 1533, 1463, 1072, 658. H NMR (400 3004, 2959-2851, 1531, 1462, 1320, 1074, 953, 841; 700, 619.
1
MHz, DMSO-d6) δ/ppm 1.65 – 2.16 (m, 5 H, H4, H5 and H8), 1H NMR (400 MHz, DMSO-d6) δ/ppm 1.84 – 2.34 (m, 5 H,
3.03 - 3.08 (m, 1 H, H2b), 3.20 - 3.45 (m, 4 H, H6 and H7), H4, H5 and H8), 3.27 - 3.67 (m, 5 H, H2a, H6 and H7), 3.96 -
3.60 – 3.68 (m, 1 H, H2a), 4.04 - 4.08 (m, 1 H, H3), 4.40 - 4.52 4.04 (m, 1 H, H2b), 4.54 (s, 2 H, CH2-Bn), 4.67 - 4.75 (m, 1 H,
(m, 2 H, CH2-Bn), 5.55 (d, J=3.18 Hz, 1 H, OH), 7.44 - 7.51 (m, H3), 7.47 - 7.52 (m, 2 H, H3’ and H5’), 7.71 - 7.76 (m, 2 H, H2’
2 H, H2’ and H6’), 7.69 - 7.77 (m, 2 H, H3’ and H5’); 13C NMR and H6’); 13C NMR (101 MHz, DMSO-d6) δ/ppm 18.71 (C5),
(101 MHz, DMSO-d6) δ/ppm 17.79 (C5), 21.34 (C8), 27.22 (C4), 22.76 (C8), 28.35 (C3), 52.35 (C6), 54.05 (C3), 54.29 (C7), 61.99
53.06 (C6), 54.33 (C7), 63.07 (C2), 63.75 (C3), 65.53 (CH2-Bn), (C2), 65.41 (CH2-Bn), 124.67 (C1’), 127.06 (C4’), 132.49 (C2’
124.50 (C1’), 127.42 (C4’), 132.41 (C2’ and C6’), 135.60 (C3’ and C6’), 135.65 (C3’ and C5’). Anal. calcd. for C14H18Br2ClN:
and C5’). Anal. calcd. for C14H19Br2NO: C 44.59; H 5.08; Br C 42.51; H 4.59; Br 40.40; Cl 8.96; N 3.54. Found: C 42.52, H
42.38; N 3.71; O 4.24. Found: C 44.58, H 5.08, N 3.71.
4.60, N 3.54.
N-p-chlorobenzyl-3-chloroquinuclidinium bromide
N-p-chlorobenzyl-3-hydroxyquinuclidinium bromide
(QOH-3). Yield: 84.0%; mp: 220.8-222.3°C; IR (KBr) v/cm-1: (QCl-3). Yield: 64.5%; mp: 186.7-188.3°C; IR (KBr) v/cm-1:
3512-3132, 3002, 2941, 1536, 1466, 1131, 1094, 1041, 951, 3003, 2958-2852, 1530, 1460, 1322, 1092, 951, 844; 700, 618.
652.1H NMR (400 MHz, DMSO-d6) δ/ppm 1.69 – 2.16 (m, 5 1H NMR (400 MHz, DMSO-d6) δ/ppm 1.82 – 2.36 (m, 5 H,
H, H4, H5 and H8), 3.02 – 3.10 (m, 1 H, H2b), 3.21 - 3.44 (m, H4, H5 and H8), 3.23 - 3.67 (m, 5 H, H2a, H6 and H7), 3.96 –
4 H, H6 and H7), 3.62 – 3.69 (m, 1 H, H2a), 4.02 – 4.09 (m, 4.05 (m, 1 H, H2b), 4.56 (s, 2 H, CH2-Bn), 4.67 - 4.75 (m, 1 H,
1 H, H3), 4.44 - 4.55 (m, 2 H, CH2), 5.55 (d, J=3.67 Hz, 1 H, H3), 7.53 - 7.64 (m, 4 H, Ar);13C NMR (101 MHz, DMSO-d6) δ/
OH) 7.53 - 7.64 (m, 4 H, Ar);13C NMR (101 MHz, DMSO-d6) δ/ ppm 18.71 (C5), 22.75 (C8), 28.34 (C4), 52.36 (C6), 54.05 (C3),
ppm 17.79 (C5), 21.34 (C8), 27.23 (C4), 53.05 (C6), 54.29 (C7), 54.27 (C7), 61.99 (CH2-Bn), 65.35 (C2), 126.67 (C1’), 129.55
63.05 (CH2-Bn), 63.74 (C3), 65.39 (C2), 127.07 (C1’), 129.46 (C2’ and C6’), 135.40 (C3’ and C5’), 135.81 (C4’). Anal. calcd.
(C2’ and C6’), 135.35 (C3’ and C5’), 135.65 (C4’). Anal. calcd. for C14H18BrCl2N: C 47.89; H 5.17; Br 22.76; Cl 20.19; N 3.99.
for C14H19ClBrNO: C 50.55; H 5.76; Br 24.02; Cl 10.66; N 4.21; Found: C 47.88, H 5.17, N 3.99.
O 4.81. Found: C 50.54, H 5.76, N 4.22.
N-p-nitrobenzyl-3-chloroquinuclidinium
bromide
N-p-nitrobenzyl-3-hydroxyquinuclidinium bromide (QCl-4). Yield: 99.0%; mp: 177.4-179.0°C; IR (KBr) v/cm-1:
(QOH-4). Yield: 95.1%; mp: 210.4-210.3°C; IR (KBr) v/cm-1: 3012, 2955-2847, 1559, 1473, 1348, 1092, 950, 870; 701, 622.
3510-3140, 3003, 2940, 1530, 1465, 1120, 1042, 1010, 952. 1H 1H NMR (400 MHz, DMSO-d6) δ/ppm 1.82 – 2.35 (m, 5 H,
NMR (400 MHz, DMSO-d6) δ/ppm 1.68 – 2.17 (m, 5 H, H4, H4, H5 and H8), 3.32 - 3.69 (m, 5 H, H2a, H6 and H7), 4.00 -
H5 and H8), 3.07 – 3.14 (m, 1 H, H2b), 3.25 - 3.49 (m, 4 H, H6 4.09 (m, 1 H, H2b), 4.68 - 4.76 (m, 3 H, CH2-Bn and H3), 7.83
and H7), 3.66 - 3.71 (m, 1 H, H2a), 4.04 - 4.08 (m, 1 H, H3), - 7.87 (m, 2 H, H2’ and H6’), 8.32 - 8.41 (m, 2 H, H3’ and H5’);
4.60 - 4.72 (m, 2 H, CH2-Bn), 5.57 (d, J=3.18 Hz, 1 H, OH), 13C NMR (101 MHz, DMSO-d6) δ/ppm 18.73 (C5), 22.77(C8),
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28.25 (C4), 52.67 (C6), 54.00 (C3), 54.56 (C7), 62.20 (C2), (Rockville. MD. USA) and at the Microbiology laboratory.
64.86 (CH2-Bn), 124.33 (C2’ and C6’), 134.80 (C1’), 135.17 Department of Biology, Faculty of Science, University of
(C3’ and C5’), 149.11 (C4’). Anal. calcd. for C14H18BrClN2O2: Split, Croatia (FNSST). The assayed collection included
C 46.49; H 5.02; Br 22.09; Cl 9.80; N 7.75; O 8.85. Found: C three Gram-positive bacteria Bacillus cereus (ATCC 11778),
46.50, H 5.02, N 7.74.
Enterococcus faecalis (ATCC 29212), Staphylococcus aureus
(ATCC 25923) and three Gram-negative ampicillin-resistant
bacterialstrainsEscherichiacoli(FNSST111),Pseudomonas
aeruginosa (FNSST 982) and Cronobacter (Enterobacter)
sakazakii (FNSST 014). Bacterial strains were cultured
overnight at 37°C in tryptic soy broth (TSB) and fungi were
cultured overnight at 30°C in Sabouraud dextrose broth
2.3 In vitro antioxidant activity
2.3.1 Oxygen radical absorbance capacity (ORAC) assay
The antioxidant activities were determined by ORAC (SDB) to achieve optical densities corresponding to 106
method [26], which measures the decay in fluorescence colony forming units (cfu/mL) for bacteria and 104 cfu/mL
intensity (FI) of a probe in time. Prior to the experiment, all for fungal strains. Antifungal activity was assessed on the
chemicals were dissolved in phosphate buffer (0.075 mM, yeast Candida albicans (ATCC 10231) and fungal strains
pH 7.0). The FI decay of the reaction mixture containing 190 Penicillium notatum (FNSST 3729) and Aspergillus niger
µL fluorescein (160 µM), 60 µL AAPH (150 mM) and 30 µL (FNSST 3843).
of the tested sample or a reference standard Trolox (6.25-50
µM) was measured in a microtiter plate (Porvair Sciences)
at 37°C in a Perkin-Elmer LS55 spectrofluorimeter using 2.4.2 Disc diffusion assay
the excitation wavelength of 485 nm. The fluorescence
decay was monitored at 530 nm during the period of 60 In order to investigate the antimicrobial activities of
min. The data were analyzed using FW WinLab sofware the novel synthesized ammonium salts containing
and the obtained results represent an average of at least quinuclidine moiety a disc diffusion assay was employed
three independent experiments.
according to the CLSI guidelines [CLSI. 2008]. Briefly, 100
µL of suspension containing 106 colony-forming units (cfu/
mL) of bacterial cells were spread on a Mueller Hinton
agar (Becton Dickinson, Sparks, MD). The stock solutions
of quaternary salts were prepared by dissolving in 96%
2.3.2 DNA protection assay
The DNA protection assay was performed in a reaction ethanol to a final concentration of 10 mg mL-1. The sterile
mixture containing supercoiled pQE60 plasmid (200 ng) filter discs (6 mm) were individually loaded with 40 µL of
and different concentrations of the QOH-4 compound the stock solution equivalent to a final concentration of
(0-100mM)orcatalase(0,2Uand0,5U)asapositivecontrol. 400 µg/disc of synthesized compounds and then placed on
Afer a 10 min incubation period at room temperature, in the nutrient agar that had been previously inoculated with
the reaction mixture 10 µL Fenton’s reagent (30 mM H2O2, the target microbial strains. Additionally, 96% ethanol was
50 µM ascorbic acid and 80 µM of FeCl3) was added. The used as a negative control cefotaxime (30 μg) gentamicin
reaction mixture was incubated for another 30 min at 37°C (15 μg) and commercial fungicide amphotericin B (10 μg)
following detection using the ethidium bromide (0.5 µg were used as positive controls. The plates were incubated
mL-1) in a 1% agarose gel. The DNA bands were separated for 24 h at 37°C for bacterial strains and 48 h or 72 h at 30°C
by applying the electric current of 75 mA for 30 min in the for yeast and mold isolates respectively. Antibacterial
standard TAE buffer (40 mM Tris, pH 7.6; 20 mM acetic acid activity was assessed by measuring the diameter of the
and 1 mM EDTA) and visualized using the ChemiDock XRS inhibition zone in millimeters including disc diameter for
imaging system (Bio-Rad).
the test isolates compared to the controls. Samples were
assayed in triplicate for each condition and the diameter
of inhibition zones were presented as mean SE values.
2.4 Biological assays
2.4.1 Assay for in vitro antimicrobial activity
2.4.3 Minimal inhibitory concentration assay
The tested microorganisms were obtained from the culture Inadditionantimicrobialactivitiesofthenovelsynthesized
collection at the American Type Culture Collection (ATCC) ammonium salts containing quinuclidine moiety were
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tested by a broth microdilution assay in 96 well microtitre 3-chloroquinuclidine quaternized with benzyl or para-
plates. The standard two fold serial microdilution assay bromo, para-chloro and para-nitrobenzyl bromides
described by the Clinical and Laboratory Standards (Scheme 1). All synthesized products were of
a
Institute [CLSI. 2008] was performed for the assessment satisfactory purity and therefore no further purification
of the minimal inhibitory concentrations (MICs). Bacteria by recrystallization was necessary. The white crystals of
and fungi were grown overnight in Mueller-Hilton broth quaternary salts were obtained in very good yields. The
1
(MHB) at 37°C and Sabouraud dextrose broth (SDB) at acquired H- and 13C-NMR signals of the quinuclidine
30°C to the stationary phase. The microbial cultures were moiety were in accordance with the previously published
diluted in fresh MHB and SDB to a final concentration of data [24, 25]. The assignment of the aromatic benzyl
106 cfu/mL for bacteria. The compounds in the aqueous rings was based on the NMR signals of chemical shifs
nutrient medium were previously dissolved in 96 % and multiplicity of 1H-NMR. The attributions of the
1
ethanol to the highest concentration of 10 mg mL-1. aromatic H-NMR chemical shifs are in full agreement
The stock solution was then serially two fold diluted to with those previously reported for the N-benzyl-3-
obtain concentrations ranging from 0.06-1024 µg mL-1 in benzamidoquinuclidines except the signal of H-4, which
sterile plates containing Mueller Hinton broth (MHB) for is missing due to the para-substitution. On the other hand,
13
bacteria. Serial dilutions of the investigated compounds the aromatic C–signals are not in a correlation with the
were added to the microtiter plates in a volume of 100 previously obtained N-benzyl-3-benzamidoquinuclidine
µL. Each well was additionally inoculated with 100 µL spectral data, instead somewhat higher 13C-chemical shifs
of inoculums of the target microorganism and incubated of C-3, C-4 and C-5 atoms in the benzyl rings are observed
at 37°C for 18-24 h. The MIC value was determined as the and it is most likely due to the effect of a substituent.
lowest concentration of the sample at which the tested
microorganisms did not demonstrate any visible growth
afer incubation. As an indicator of bacterial growth, 50 3.2 In vitro Antioxidant activity
μL of 0.2 mg mL-1 p-iodonitrotetrazoliumchloride (INT;
Sigma-Aldrich Co. Ltd, Poole, UK) was added to the wells 3.2.1 ORAC assay
and incubated at 37°C for 30 min. Following addition of
INT and incubation the MIC was determined as the lowest The observed biological activity of some quinuclidinium
sample concentration at which no pink colour appeared. salts [23] could be, among others, associated with the
Cefotaxime, gentamicin and amphotericin B were used antioxidative potential that has not been investigated so
as positive controls. Minimal inhibitory concentrations of far. In order to determine the antioxidative potential of the
the commercial antibiotics were determined by the E-test newly synthetized compounds, a standard ORAC method
(AB Biodisk, Solna, Sweden). The MIC was interpreted as was used [26,27]. This method measures the decay in
the point of intersection of the inhibition ellipse with the fluorescence intensity (FI) of a probe upon the reaction
E-test strip edge. In this study no bioactivity was defined with the free peroxyl radicals generated by AAPH. In
as a MIC >1000 µg/mL, mild bioactivity as a MIC in the the presence of the standard (Trolox), the FI decay is
range 512-1000 µg mL-1, moderate bioactivity as a MIC in postponed until the antioxidative agent is fully expended.
the range 128-512 µg mL-1, good bioactivity as a MIC in From the resultant spectra the area under the curve (AUC)
the range 32-128 µg mL-1, strong bioactivity as a MIC in is obtained afer the blank (Blk) AUC subtraction, and
the range 10-32 µg mL-1, and very strong bioactivity as a the standard curve is generated by plotting the AUC with
MIC<10 µg mL-1.
nmol of the Trolox standard. The obtained antioxidative
Ethical approval: The conducted research is not potential expressed in nmol of trolox eqvivalents (TE) is
related to either human or animals use.
presented in Table 1.
The observed data show that all quaternary
quinuclidinium compounds exhibit better antioxidative
activity than the precursor compounds (QOH and QCl).
Among the tested quaternary quinuclidinium salts,
compounds QOH-2 and QOH-4 exhibit the highest
antioxidative potential. Therefore, it can be concluded
that the hydroxy group at the position 3 on quinuclidine
3 Results and discussion
3.1 Synthesis
Two series of quaternary ammonium salts (QAS) have ring has a significant influence on the antioxidative
been synthesized by the reaction of 3-hydroxy or activity. This finding might be explained by the polarity
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R
+
N
N
OH
OH
Br-
acetone
R
QOH-1-4
CH2Br
+
QOH
R
R
KOH
N
Cl
HCl
N
Cl
-H
-Br
-Cl
-NO2
1
2
3
4
+
N
Cl
Br-
QCl
QCl-1-4
Scheme 1: Synthesis of quaternary ammonium salts.
Table 1: Antioxidative activity of the tested compounds, determined
produced base radicals further interact with sugar moiety,
causing the breakage of the sugar-phosphate backbone
and linearization of the DNA molecule. The resultant DNA
molecule is visualized by gel electrophoresis whereby
linear and circular DNA forms are easily distinguished.
The results of ORAC assay have shown that among tested
compounds, QOH-4 have the strongest antioxidative
potential. In order to further characterize this finding
we have performed the DNA protection assay aiming to
evaluate the capacity of QOH-4 to prevent linearization of
pQE60 plasmid upon the addition of the Fenton’s reagent.
Figure 1. shows the protective effect of QOH-4 and its
ability to shield DNA from the Fe3+-dependent nicking.
When DNA was exposed to Fenton’s reagent it resulted
in the formation of the linear (nicked) DNA form. Addition
by ORAC method.
Compound
QOH
nmol (TE) mL-1
22.85 0.45
QCl
QOH-1
21.36 0.64
40.09 0.24
QCl-1
51.24 0.32
111.6 0.38
QOH-2
QCl-2
QOH-3
QCl-3
QOH-4
QCl-4
57.43 0.29
27.21 0.19
63.72 0.25
293.80 0.85
88.40 0.12
of the hydroxyl group which could act as a proton donor of QOH-4 resulted in a partial preservation of the circular
in the redox reaction and thereby diminish the influence DNA, which was shown to be concentration dependent.
of the peroxyl radicals. Moreover, it appears that in the These results suggest that QOH-4 have the ability to shield
series of the QOH compounds, the NO2 substituent at DNA in an oxidative stress condition which might be
para position on the benzyl ring strongly influences explained either by the QOH-4-iron complex formation or
antioxidative activity. This was also observed with the by a proton donor ability of the hydroxyl-group. Hydroxyl-
QCl series, which albeit of generally lower antioxidative groups are known to have a reducing potential and are
potential, detain similar influence of the NO2 substituent able to reduce highly oxidizing free radicals such as
on the antioxidative activity.
superoxide, peroxyl, aloxyl and hydroxyl radicals [28].
3.2.2 DNA protection assay
3.2.3 Biological evaluation
The assay is based on the ability of the tested sample 3.2.3.4 Disc diffusion assay
to neutralize free radicals and prevent breaking the In this study the preliminary antimicrobial potential of
DNA strands. The DNA damage is induced by Fenton’s the N-quaternary salts was tested against a diverse panel
reagent, which serves as a generator of the hydroxyl (●OH) of selected antibiotic susceptible Gram-positive bacteria
radicals that react with the nitrogen bases of the DNA. The including Bacillus cereus (ATCC 11778), Enterococcus
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326ꢀ
ꢀ Renata Odžak et al.
faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), resistance profiles. Among the tested Gram-negative
Gram-negative ampicillin-resistant bacteria namely bacteria, Pseudomonas aeruginosa displayed promising
Escherichia coli (FNSST 111), Pseudomonas aeruginosa sensitivity towards all compounds with considerable
(FNSST 982) and Cronobacter (Enterobacter) sakazakii zones of growth inhibition which were larger than the
(FNSST 014) and fungi such as Candida albicans (ATCC ones with the reference positive controls, GEN and CTX
10231) Penicillium notatum (FNSST 3729) and Aspergillus respectively (Figure 2 A).
niger (FNSST 3843) by the disc diffusion assay.
This is important, because P. aeruginosa is a major
As a comparison, we tested the inhibition zones of opportunistic pathogen that causes a wide range of
conventional antimicrobial agents that are used in clinical nosocomialinfectionsandaccumulatedenoughresistance
settings to treat infections caused by pathogenic bacteria, mechanisms to render them virtually untreatable by
such as gentamicin (GEN) and cefotaxime (CTX). The modern antibacterial chemotherapy [29].
antifungal agent amphotericin B (AMPHB), was used as a
referent antimicotic.
Promising antimicrobial potential of all new
compounds was also observed against E. coli but only
As shown in Table 2 the results of the disc diffusion in comparison to GEN (Figure 2 B). CTX was still more
assay indicate that all compounds exhibit a potent and efficient for that bacterium, displaying a larger zone of
broad spectrum activity against tested bacterial and growth inhibition than either of the compounds. It is
fungal strains.
worth noting that among the tested quinuclidinium salts
The mean zones of inhibition were found to be in the QCl-4 displayed a potential antimicrobial activity against
range of 9.3 0.3 to 24.3 1.3 mm. It is noteworthy that the both, bacteria and fungi (Figure 3). However, it should be
tested samples displayed not only antibacterial activity noted that the amount of tested quinuclidines was 20 folds
against the susceptible Gram-positive bacteria but also higher than the reference antibiotics, thus more detailed
against Gram-negative bacteria with various antibiotic evaluation of antimicrobial potential was further needed.
3.2.4 Minimal inhibitory concentrations (MICs)
The antimicrobial activity was investigated by a broth
microdilution method to determine the minimal inhibitory
concentrations (MICs) for two series of quaternary
quinuclidinium salts. MIC is the minimal concentration of
the tested compound at which inhibition of the bacterial
growth is observed. As a control, we have also tested MICs
of the relevant conventional antimicrobial agents such as
gentamicin and cefotaxime which are currently used in
clinical treatments. The results presented in Table 3 reveal
that all compounds display a remarkable selectivity and
Figure 1: DNA protection assay of QOH-4. The pQE60 plasmid in
the presence and absence of the DNA nicking reagent (Fenton’s
reagent). Catalase was used as a positive control.
A)
B)
Figure 2: Diameters of the inhibition zones of all tested quinuclidinium salts against P. aeruginosa (A) and E. coli (B). Reference antibiotics
GEN and CTX (A) and GEN (B) served as positive controls.
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328ꢀ
ꢀ Renata Odžak et al.
that different para substituents at benzyl rings of
quaternary salts affects antibacterial activity as accounted
for 3-chloroquinuclidines bearing Br substituent on the
benzyl ring. For these compounds strong activity against
Gram-negative bacteria was observed. On the other hand,
quinuclidiniumsaltscontainingNO2groupasasubstituent
on the benzyl group, show strong antibacterial activity
against all tested Gram-positive bacteria. It should also be
noted that Cl as a para substituent on the benzyl group
positively affects antimicrobial activity against respective
Gram-positive and Gram-negative bacteria, regardless
if it is part of a 3-hydroxy or 3- chloroquinuclidinium
Figure 3: Antibacterial and antifungal activities of QCl-4 against
all tested microorganism in comparison with GEN and AMPHB as
positive controls.
effective activity against the tested Gram-positive and heterocyclic core.
Gram-negative bacteria with MIC values ranging from
The obtained MIC values for all compounds against
0.39 to 100.00 μg mL-1. Detailed analysis of the obtained three fungal strains are presented in Table 4. Our results
MIC values show that quaternary salts with substituents show better antifungal activities for C. albicans with the
at para position such as Br, Cl and NO2, display generally compounds from the QOH series. The most prominent MIC
better activity than the compounds without substituents values were observed for QOH-4 (MIC=1.56 μg mL-1), which
on the benzyl ring regardless of the substituent type. is in range with the AMPHB standard.
However, the exception to this observation are data for P.
aeruginosa which for all tested compounds had lower MICs
then the standard antibiotic GEN and for most compounds 4 Conclusions
displayed lower MICs than the reference CTX.
Among the eight newly synthetized quinuclidinium In this study, we have synthesized a novel series of
salts, two compounds namely QOH-3 and QCl-3 displayed N-quaternarysaltsof3-hydroxy-and3-chloroquinuclidines
better antibacterial potential against the Gram-positive (B. (QOH-1-4 and QCl-1-4) to investigate their antimicrobial
cereus and E. faecalis) and Gram-negative bacteria (E. coli and antioxidative activities. All compounds were
and P. aeruginosa) than the reference antibiotics with MIC synthesized and characterized by spectral data (IR, 1D
values ranging from 0.39-3.12 μg mL-1. QCl-3 had 2.5-fold and 2D NMR). The ORAC test was used to evaluate the
better antibacterial activity than GEN against B. cereus antioxidant properties of synthesized QAS. According
and QOH-3 had similar MIC value against E. faecalis as to our ORAC results and the DNA protection assay the
GEN. For E. coli MIC value observed in the presence of QOH-4 compound was identified as the most potent
the QCl-3 is 10-fold lower than the reference GEN. These antioxidants. In preliminary screening studies, the
values for QOH-3 are even 164-fold lower then GEN and obtained antimicrobial results were very promising since
41-fold lower than CTX.
many of the compounds demonstrated a potent in vitro
Strong and potent antimicrobial activity for other activity against a broad range of clinically important Gram-
quinuclidinium compounds have also been observed. For negative bacteria, including multidrug resistant strains
example, QCl-4 showed identical MIC values (6.25 μg mL-1) with MIC value from 0.39 to 3.12 μg mL-1 (QCl-2, QOH-2,
for all tested Gram-positive bacteria which is comparable QCl-3 and QOH-4). Some of them, such as QCl-4, showed
to the GEN. Furthermore, QCl-2 and QOH-4 display MIC very strong bioactivity against all tested Gram-positive
values for P. aeruginosa (3.12 μg mL-1) which are 20-fold bacteria with MIC values of 6.25 μg mL-1 Furthermore, we
lower than the MIC observed for GEN and 5-fold lower discovered that among the present class of compounds,
than the one observed for CTX. Lower MIC values ranging QOH-3 and QCl-3, with Cl as the para-substituent on the
from 3.12-6.25 μg mL-1 have been accounted for all tested benzyl group, were especially effective against Gram-
Gram negative bacteria in the presence of QCl-2 relative positive and Gram-negative species with much lower MIC
to GEN. For C. sakazakii MIC for QCl-2 was 2.5-fold lower values than standard antibiotics tested in this study (for
than for GEN and CTX.
example, 164 fold lower than GEN) which could serve as
In conclusion, among the synthesized and tested a guidance to design new and promising antibacterial
quinuclidinium salts, QCl-3 and QOH-3 showed compounds.
promising inhibitory profiles against clinically important
pathogens such as E. coli and P. aeruginosa. It appears strong antioxidant activity and a high antifungal potential
Of special importance is the finding that QOH-4 has a
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Table 3: Minimal inhibitory concentration of 3-substituted quinuclidine (QOH and QCl) and their quaternary salts (QOH-1-4 and QCl-1-4)
against Gram-positive and Gram-negative bacteria. The MIC values lower than the reference antibiotic are indicated as bold numbers in dark
grey squares and MIC values between the two referent antibiotics are colored in grey.
Comp/Mr Structure
MIC (µg mL-1)
Gram-positive bacteria
Gram-negative bacteria
B. cereus
E. faecalis
S. aureus
E. coli
P. aeruginosa
C. sakazakii
HO
QOH
127.18
25.00
50.00
25.00
50.00
25.00
50.00
N
Cl
QCl
145.63
50.00
25.00
12.50
12.50
50.00
25.00
N
HO
QOH-1
12.50
25.00
50.00
25.00
50.00
25.00
50.00
25.00
6.25
12.50
25.00
298.22
+
N
-
Br
Cl
QCl-1
316.66
25.00
+
N
-
Br
Br
QOH-2
50.00
12.50
100.00
1.56
50.00
6.25
25.00
12.50
12.50
6.25
12.50
3.12
12.50
3.12
25.00
3.12
HO
377.11
+
N
-
Br
Br
QCl-2
395.56
Cl
+
N
-
Br
Cl
QOH-3
332.66
3.12
100.00
25.00
0.39
25.00
25.00
HO
+
N
-
Br
Cl
QCl-3
351.11
25.00
25.00
Cl
+
N
-
Br
NO2
QOH-4
50.00
6.25
12.50
6.25
100.00
6.25
25.50
6.25
3.12
50.00
25.00
HO
343.22
+
N
-
Br
NO2
QCl-4
361.66
25.00
Cl
+
N
-
Br
Gentamicin
Cefotaxime
4.00
0.25
4.00
0.50
1.00
0.50
32.00
0.50
64.00
16.00
8.00
8.00
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330ꢀ
ꢀ Renata Odžak et al.
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antimycotic is indicated in grey.
Compound
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againstC.albicanswhencomparedtootherQASderivatives
(1.59 μg mL-1). It is also worth mentioning that reference
compounds QOH and QCl showed much lower values
in all applied tests than the corresponding quaternary
salts indicating that quaternization have improved
biological potential of 3-substituted quinuclidine. The
results of the present study are suggesting a promising
starting point for ammonium salts structure optimization
based on quinuclidine scaffold in order to design potent
antimicrobial and antioxidative agents. Moreover, the
mechanism of antibacterial activity and identification
of the bacterial enzymes as potential targets of newly
synthesized compounds is of a great interest in the
future.
Acknowledgments: This work was supported by the
Croatian Ministry of Science and Education as part of
multiannual financing intended for institutions.
Conflict of interest: Authors state no conflict of interest.
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