Antifungal activity of styrylpyridinium compounds against Candida albicans

Article abstract of DOI:10.1111/cbdd.13777

We synthesized a set of 13 new and earlier described styrylpyridinium compounds (N-alkyl styrylpyridinium salts with bromide or tosylate anions) in order to evaluate antifungal activity against C.?albicans cells, to assay the possible synergism with fluco

Full text of DOI:10.1111/cbdd.13777

DR SIMONA VAITKIENĖ (Orcid ID : 0000-0002-6550-1485)
Article type : Research Article
Antifungal activity of styrylpyridinium compounds against Candida albicans
Simona Vaitkienė1*, Neringa Kuliešienė1, Sandra Sakalauskaitė1, Laura Bekere2, Laura Krasnova2,
Brigita Vigante2, Gunars Duburs2 and Rimantas Daugelavičius1
1Department of Biochemistry, Vytautas Magnus University, Vileikos str. 8, Kaunas 44404,
Lithuania;
2Latvian Institute of Organic Synthesis, Aizkraukles str. 21, Riga 1006, Latvia
*Correspondence: Simona Vaitkienė, Department of Biochemistry, Faculty of Natural Sciences,
Vytautas Magnus University, Vileikos 8, LT-44404 Kaunas, Lithuania
E-mail: simona.vaitkiene@vdu.lt
This article has been accepted for publication and undergone full peer review but has not been
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differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13777
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Keywords:
Candida albicans
Styrylpyridinium compounds
Fungicidal activity
Cytotoxicity
Abstract
We synthesized a set of 13 new and earlier described styrylpyridinium compounds (N-
alkyl styrylpyridinium salts with bromide or tosylate anions) in order to evaluate antifungal
activity against C. albicans cells, to assay the possible synergism with fluconazole, and to estimate
cytotoxicity to mammalian cells. All compounds were synthesized according to a well-known
two-step procedure involving alkylation of γ-picoline with appropriate alkyl bromide and further
condensation with substituted benzaldehyde.
Compounds with long N-alkyl chains (C18H37–C20H41) had no antifungal activity against
the cells of all tested C. albicans strains. Other styrylpyridinium compounds were able to inhibit
yeast growth at the concentrations of 0.06 to 16 μg/ml. At fungicidal concentrations, the
compound with the CN- group was least toxic to mammalian cells, showed the most effective
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synergism with fluconazole, and only slightly inhibited the respiration of C. albicans. The
compound with the 4′-diethylamino group exhibited the strongest fungicidal properties and
effectively blocked the respiration of C. albicans cells. However, toxicity to mammalian cells was
also high.
Summarizing, the results of our study indicate that styrylpyridinium compounds are
promising candidates in the development of new antifungal drugs.
1. Introduction
Yeasts of Candida genus, especially Candida albicans (C. albicans), are the most common
causes of fungal infections. Recent studies have indicated that the frequency of invasive
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candidiasis and candidemia is increasing [1]. The management of these infections depends on the
immune status of a host, severity of a disease, and choice of antifungal drugs [2]. The leading
cause of spread and progression of fungal infections is a weakened immune system, which is
characteristic of elderly persons. The high-risk group also involves chemotherapy-treated cancer
patients, persons after organ transplantations, or HIV-infected patients treated with
immunosuppressant drugs. Moreover, the risk of being infected increases for diabetic patients,
possibly due to the impact of glucose on C. albicans metabolism [3]. The developed severe fungal
infection affects skin, mucosa and spreads to the organs and blood stream [4, 5].
The choice of medicines to treat fungal infections is limited due to emerging yeast resistance
to antifungal compounds [6]. Most frequently, multidrug resistance (MDR) evolves due to
transporters in the yeast plasma membrane that pump a broad spectrum of antimicrobial molecules
out of cells [7]. C. albicans possesses two types of efflux pumps: ATP-binding cassette (ABC)
family transporters and major facilitator superfamily (MFS) pumps that use ATP or
electrochemical proton gradient, respectively, to drive active transport of their substrates across
the plasma membrane. One of the possible ways to reduce C. albicans resistance to antimicrobials
is to use inhibitors reducing efflux activity. Inhibitors can reduce the efflux of antimicrobial agents
by inactivating pumps or competing with pump substrates [8]. Another way of fighting
resistance—usage of drug combinations— appears as an effective approach to improve the
antifungal efficiency of antimicrobials on multiple targets. Synergism of compounds leads to a
shorter duration of treatment, lower doses, and increased efficacy of single drugs [9].
Reduced fungal susceptibility to polyenes, echinocandins, and azoles warrants the search for
new antifungal agents, e.g., pyridine derivatives. N-containing heterocyclic compounds are widely
found in nature, have diverse biological activities, and affect various cellular targets. Recently,
pyridines have emerged as a substantial class of heterocycles that are endowed mainly with
anticancer activities [10]. As a result, pyridines have become an appealing target for medicinal
chemists around the world.
Styrylpyridinium compounds—lipophilic cations linked to pyridine—were initially
synthesized as fluorescent probes for studies on cell membranes in normal and pathological states
[11-13]. Nowadays, the use of such compounds is wider [14-16], but still they mainly serve as
probes for pathological studies. Studies on the activity of styrylpyridinium compounds against
Gram-positive and Gram-negative bacteria are scarce [17].
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Considering the information mentioned above, we aimed to evaluate the antifungal efficiency
of newly synthesized and earlier described styrylpyridinium compounds on C. albicans cells, to
estimate their cytotoxicity to mammalian cells, and to evaluate the synergistic antifungal effects in
combination with fluconazole.
2. Material and methods
2.1.
Synthesis of styrylpyridinium compounds
All 13 compounds (Table 1) were synthesized according to a two-step procedure involving
alkylation of γ-picoline with appropriate alkyl bromide and further condensation with substituted
benzaldehyde under conditions recently reported elsewhere [11]. The synthesis pathways of
compounds I–XIII are depicted in Scheme 1. According to the reported procedure [18], the known
1-alkyl-4-methylpyridinium bromides were obtained by the treatment of γ-picoline with alkyl
halides in acetonitrile solution at 80 °C temperature for 24 h. N-Alkyl styrylpyridinium bromides
bearing long-chain alkyl groups (C8, C10, C12, C18, and C20) at the nitrogen atom of the pyridine
moiety and different substituents in the aryl group were obtained at moderate yields by the
condensation of N-alkyl pyridinium bromides with appropriate substituted benzaldehyde in
methanol solution at reflux for 5 h. For the synthesis of compound VII, N-hexyl pyridinium
tosylate was used instead of the bromides mentioned above [13].
2.2.
Chemistry
All reagents were purchased from Acros Organics, Sigma-Aldrich, or Alfa Aesar and used
without further purification. One-dimensional 1H and 13C NMR spectra were recorded at 400 MHz
(1H) and 100 MHz (13C) operating frequency with a Varian 400 Mercury spectrometer. Chemical
shifts of the hydrogen and carbon atoms are presented in parts per million (ppm) and referred to
the residual signal of non-deuterated CDCl3 (δ:7.26) or partially deuterated D6-DMSO (δ: 2.50).
Low-resolution mass spectra (MS) were determined on an Acquity UPLC system (Waters)
connected to a Waters SQ Detector 2 operating in the ESI positive ion mode on a Waters Acquity
UPLC BEH C18 column. Elemental analyses were performed on an Elemental Combustion
System ECS 4010 (Costech Instruments) at the Laboratory of Chromatography, Latvian Institute
of Organic Synthesis. Logarithms of the partition coefficients logP were calculated by means of
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Molinspiration; logP’, topological polar surface area PSA, pKa and distribution coefficient logD
were calculated by means of Chem3D Ultra 18.2, PerkinElmer Informatics.
The identities of N-alkyl 4-methyl pyridinium bromides and Comps I–IV and VI–VIII,
which have been previously reported [11-13, 18-21, 23, 24], were confirmed by 1H NMR. Comps
1
13
V, IX-X, and XI–XIII were novel and were fully characterized by H NMR, C NMR, MS, and
elemental analysis.
General procedure for the synthesis of 4-(substituted styryl)-1-alkylpyridinium bromides
The mixture of appropriate 1-alkyl-4-methylpyridinium bromide (1 mmol, 1 eq), substituted
benzaldehyde (1 mmol, 1 eq), and piperidine (5 drops) in methanol (10 mL) was refluxed for 5 h.
The solvent was removed in vacuo and the residue was triturated with dry acetone. After cooling,
the precipitate was filtered and recrystallized from acetonitrile. All target compounds had >95%
purity. The purities of all compounds were determined by HPLC on a Waters Alliance 2695
system and Waters 2489 UV-vis detector equipped with an Altima C18 column, 5 M, 4.6 × 150
mm, using gradient elution with acetonitrile/H3PO4 (0.1%) in water at a flow rate of 1 mL/min.
(E,Z)-4-(3,4-Dimethoxystyryl)-1-dodecylpyridin-1-ium bromide (Comp V)
The title compound was synthesized according to the general procedure from 1-dodecyl-4-
methylpyridinium-1 bromide (342 mg, 1 mmol) and 3,4-dimethoxybenzaldehyde (166 mg,
1 mmol) to afford V (210 mg, 51%) as yellow powder. 1H NMR (400 MHz, CDCl3): 9.01 (d, J =
6.9 Hz, 2H), 8.03 (d, J = 6.8 Hz, 2H), 7.67 (d, J = 16.1 Hz, 1H), 7.23–7.20 (m, 2H), 7.09 (d, J =
16.1 Hz, 1H), 6.90 (d, J = 8.9 Hz, 1H), 4.72 (t, J = 7.4 Hz, 2H), 3.98 (s, 3H), 3.93 (s, 3H), 2.02–
13
1.92 (m, 2H), 1.30 (d, J = 14.1 Hz, 6H), 1.22 (s, 12H), 0.91–0.80 (m, 3H). C (CDCl3): 153.78,
151.87, 149.48, 143.93, 142.14, 127.73, 123.80, 123.67, 120.08, 111.16, 109.92, 60.73, 56.36,
56.07, 31.71, 31.65, 29.02, 29.00,26.11, 22.56, 14.04.MS (ES+) m/z:411 ([M+H-Br]+, 100).
Anal.Calcd. for C27H40BrNO2: C, 66.11; H, 8.22; N, 2.86; found: C, 65.85; H, 7.98; N, 2.76.
(E,Z)-4-(4-(Diethylamino)styryl)-1-dodecylpyridin-1-ium bromide (Comp IX)
The title compound was synthesized according to the general procedure from 4-methyl-1-dodecyl-
4-methylpyridinium-1 bromide (342 mg, 1 mmol) and 4-diethylaminobenzaldehyde (177 mg, 1
1
mmol) to afford IX (346 mg, 69%) as red powder. H NMR (400 MHz, DMSO-d6): 8.75 (d, J =
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7.6 Hz, 2H), 8.08–7.98 (m, 2H), 7.90 (d, J = 16.1 Hz, 1H), 7.60–7.51 (m, 2H), 7.60–7.51 (m, 2H),
7.17 (d, J = 16.1 Hz, 1H), 4.39 ( t, J = 7.3 Hz, 2H), 3.42 (q, J = 7.1 Hz, 4H), 1.85 (t, J = 7.1 Hz,
2H), 1.23 (s, 18H), 1.12 (t, J = 7.1 Hz, 6H), 0.92–0.71(m, 3H). 13C NMR (DMSO-d6): 154.25,
149.96, 146.36, 143.87, 142.73, 137.96, 128.49, 125.97, 122.70, 111.87, 59.54, 44.34, 32.77,
30.94, 30.46, 29.37, 29.26, 29.19, 28.86, 25.89, 22.57, 21.25, 14.43, 12.97. MS (ES+) m/z:422
([M+H-Br]+, 100). Anal. Calcd. for C29H45BrN2: C, 69.44; H, 9.04; N, 5.61; found: C, 69.15; H,
9.10; N, 5.55.
(E,Z)-1-Octadecyl-4-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-i,j]quinolin-9-yl)vinyl)pyridine-
1-ium bromide (Comp X)
The title compound was synthesized according to the general procedure from 4-methyl-1-
octadecylpyridinium-1 bromide (426 mg, 1 mmol) and 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-
1
i,j]quinoline-9-carbaldehyde (201 mg, 1 mmol) to afford X (320 mg, 50%) as red powder. H
NMR (400 MHz, CDCl3): 8.81 (d, J = 7.0 Hz, 2H), 7.74 (d, J = 7.0 Hz, 2H), 7.49 (d, J = 15.8 Hz,
1H), 7.08 (s, 2H), 6.74 (d, J = 15.8 Hz, 1H), 4.62 (t, J = 7.4 Hz, 2H), 3.29 (dd, J =6.6, 4.9 Hz, 3H),
2.75 (t, J = 6.3 Hz, 3H), 2.00–1.88 (m, 6H), 1.23 (d, J = 8.1 Hz, 32H), 0.93–0.81 (m, 3H). 13C
(CDCl3): 135.40, 147.76, 145.08, 143.68, 133.95, 129.58, 125.57, 125.04, 124.54, 123.23, 60.67,
56.00, 46.16, 31.87, 31.36, 29.61, 29.43, 29.37, 29.10 28.84, 28.70, 27.70, 26.15, 25.86,22.67,
21.30, 14.07. MS (ES+) m/z:532 ([M+H-Br]+, 100). Anal. Calcd. for C37H57 BrN2: C, 72.88; H,
9.42; N, 4.59; found: C, 72.51; H, 9.21; N, 4.35.
(E,Z)-4-(4-Cyanostyryl)-1-dodecylpyridin-1-ium bromide (Comp XI)
The title compound was synthesized according to the general procedure from 4-methyl-1-
dodecyl-4-methylpyridinium-1 bromide (342 mg, 1 mmol) and 4-cyanobenzaldehyde (131 mg, 1
mmol) to afford XI (270 mg, 59%) as red powder. 1H NMR (400 MHz, DMSO-d6): 9.07–8.99 (m,
2H), 8.32–8.24 (m, 2H), 8.08 (d, J = 16.4 Hz, 1H), 7.97 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.5 Hz,
2H), 7.72 (d, J = 16.4 Hz, 1H), 4.52 (t, J = 7.4 Hz, 2H), 1.96–1.85 (m, 2H), 1.23 (s, 18H), 0.89–
0.78 (m, 3H). 13C NMR (CDCl3): 153.80, 151.77, 149.43, 143.90, 142.14, 127.70, 123.82, 123.60,
120.10, 111.16, 110.50, 109.80, 60.72, 56.37, 56.07, 31.71, 31.64, 29.02, 29.00, 26.11, 22.55,
14.04. MS (ES+) m/z:376 ([M+H-Br]+, 100). Anal. Calcd. for C26H35BrN2: C, 68.56; H, 7.75; N,
6.15; found: C, 68.12; H, 7.65; N, 6.05.
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(E,Z)-4-(2-([1,1`-Biphenyl]-4-yl)vinyl)-1-octadecylpyridin-1-ium bromide (Comp XII)
The title compound was synthesized according to the general procedure from 4-methyl-1-
octadecylpyridinium-1 bromide (426 mg, 1 mmol) and [1,1′ -biphenyl]-4-carbaldehyde (182 mg, 1
1
mmol) to afford XII (300 mg, 51%) as yellow powder. H NMR (400 MHz, DMSO-d6): 8.98 (d,
J = 6.9 Hz, 2H), 8.30-8.24 (m, 2H), 8.09 (d, J = 16.4 Hz, 1H), 7.88–7.80 (m, 4H), 7.78–7.73 (m,
2H), 7.60 (d, J = 16.4 Hz, 1H), 7.50 (dd, J = 8.3, 6.8 Hz, 2H), 7.44–7.38 (m, 1H), 4.50 (t, J = 7.3
Hz, 2H), 2.91 (t, J = 7.1 Hz, 2H), 1.22 (s, 30H), 0.90–0.76 (m, 3H).13C NMR (DMSO-d6): 160.38,
144.78, 144.12, 140.86, 139.80, 139.07, 137.46, 130.54, 129.54, 129.45, 129.36, 129.32, 129.07,
127.95, 127.75, 127.70, 127.18, 126.86, 126.84, 124.35, 60.31, 46.16, 31.76, 31.00, 30.90, 29.51,
29.47, 29.37, 29.17, 28.84, 28.70, 25.86, 22.56, 14.42. MS (ES+) m/z:511 ([M+H-Br]+, 100). Anal.
Calcd. for C37H52BrN: C, 75.23; H, 8.87; N, 2.37; found: C, 74.84; H, 8.88; N, 2.27.
(E,Z)-4-(2-([1,1′-Biphenyl]-4-yl)vinyl)-1-icosylpyridin-1-ium bromide (Comp XIII)
The title compound was synthesized according to the general procedure from 1-icosyl-4-
methylpyridinium-1 bromide (440 mg, 1 mmol) and [1,1′-biphenyl]-4-carbaldehyde (182 mg,
1
1 mmol) to afford XIII (350 mg, 56%) as yellow powder. H NMR (400 MHz, DMSO-d6): 8.98
(d, J = 6.5 Hz, 2H), 8.30–8.23 (m, 2H), 8.09 (d, J = 16.3 Hz, 1H), 7.88–7.9 (m, 4H), 7.80–7.71 (m,
2H), 7.59 (d, J = 16.3 Hz, 1H), 7.50 (dd, J = 8.4, 6.9 Hz, 2H), 7.45–7.38 (m, 1H), 4.50 (t, J = 7.4
Hz, 2H), 1.96–1.84 (m, 2H), 1.22 (s, 34H), 0.89–0.77 (m, 3H). 13C NMR (CDCl3): 153.39, 144.21,
143.53, 141.67, 139.71, 133.52, 129.08, 128.99, 128.80, 128.10, 127.67, 126.99, 126.84, 124.30,
122.08, 60.98, 31.92, 31.72, 29.72, 29.67, 29.63, 29.61, 29.54, 29.41, 29.36, 29.11, 26.14, 22.69,
14.13. MS (ES+) m/z:539 ([M+H-Br]+, 100). Anal. Calcd. for C39H56 BrN: C, 75.70; H, 9.12; N,
2.26; found: C, 73.15; H, 9.18; N, 2.11.
2.3.
Strains, cell lines, and media
The susceptibility of C. albicans wild-type (wt) strain ATCC10231, clinical isolate strain SV1,
isolated from patients’ ascitic fluid, and DSY448 (cdr1Δ), DSY653 (cdr2Δ), DSY465 (mdr1Δ),
and DSY654 (cdr1Δcdr2Δ) mutant strains (Table 2) was tested against 13 styrylpyridinium
compounds. The mutant cells were selected to check the relationship between the compounds and
the activity of efflux pumps responsible for MDR.
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C. albicans mutant strains used in this study were a generous gift by Prof. Dominique
Sanglard (Institute of Microbiology, University of Lausanne and University Hospital Center,
Lausanne, Switzerland). The clinical isolate strain SV1 was gifted from the Laboratory of
Microbiology, Republican Panevėžys Hospital, Lithuania.
2.4.
Gene expression analysis
Expression of CDR1, CDR2, and MDR1 genes was evaluated to estimate the characteristics of
clinical isolate strain SV1. Yeast cultures were grown for 18 h in an YPD medium, and pellets
were collected by centrifugation at 4 °C for 10 min at 3000g (Heraeus Megafuge 16R,
ThermoFisher Scientific, Germany). Total RNA from SV1 and ATCC10231 cells was extracted
using a Trizol Plus RNA kit (Invitrogen, USA) with ZR BashingBead Lysis Tubes (Zymo
Research, Irvine, CA, USA). cDNA synthesis was performed using a high capacity cDNA reverse
transcription kit (Applied Biosystems, USA), according to the manufacturer’s instructions. RT-
PCR was performed using StepOneTM and StepOnePlusTM Systems (Applied Biosystems, USA) in
20-µl reaction volume consisting of Power SYBR Green RT-PCR Mix (ThermoFisher Scientific),
cDNA template, and forward and reverse primers (ThermoFisher Scientific). RT-PCR conditions
for CDR1, CDR2, and MDR1 were as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for
15 s, 60 °C for 30 s. To determine the level of CDR1, CDR2, and MDR1 expression, the
differences (∆) between the threshold cycles (Ct) were measured. Data are presented as the fold
change in gene expression normalized to the ACT1 gene as a control.
The following primers were used in the study:
CDR1: 5′-GTACTATCCATCAACCATCAGCACTT-3′ (forward)
5′ GCCGTTCTTCCACCTTTTTGTA-3′ (reverse);
CDR2: 5′-TGCTGAACCGACAGACTCAGTT-3′ (forward)
5′- AAGAGATTGCCAATTGTCCCATA -3′ (reverse);
MDR1: 5′-TCAGTCCGATGTCAGAAAATGC-3′ (forward)
5′- GCAGTGGGAATTTGTAGTATGACAA-3′ (reverse);
ACT1 (house-keeping gene and for confirmation of the PCR process):
5′-GCTTTTGGTGTTTGACGAGTTTCT-3′ (forward)
5′-GTGAGCCGGGAAATCTGTATAGTC-3′ (reverse).
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2.5.
Susceptibility testing
The effects of 13 synthesized styrylpyridinium compounds on the growth of C. albicans cells
were assayed according to the specifications of the European Committee on Antimicrobial
Susceptibility Testing (EUCAST E.DEF 7.3). Yeast cultures were grown for 18 h in an YPD
medium, and suspensions with a final cell concentration of 1.0 × 103 CFU/ml were prepared in a
modified RPMI-1640 medium, supplemented with 2% glucose and L-glutamine, without sodium
bicarbonate, and buffered to pH 7.0 with 0.165 M 4-morpholinepropanesulfonic acid (MOPS,
Sigma–Aldrich, GmbH, Steinheim, Germany). Yeast suspensions with the serially diluted
compounds at concentrations varying from 0.25 to 16 µg/ml and serially diluted fluconazole at
concentrations varying from 0.0625 to 4 µg/ml (total volume of 100 μl), were incubated at 37 °C
for 24 h without agitation. To assess the growth, ODs of yeast suspensions were measured
spectrophotometrically at 612 nm using the TECAN GENios Pro™ plate reader. The growth of
yeast cultures in the absence of compounds was used as a control. The results are presented as the
percentage of growth relative to control samples, and MIC50 and MIC90 values are given as the
lowest concentration inhibiting 50% and 90% of growth.
2.6.
Susceptibility testing of styrylpyridinium compounds in combination with fluconazole
Interactions between fluconazole and the tested compounds were evaluated by determining the
fractional inhibitory concentration (ΣFIC) index values [27]. C. albicans cells were grown the
same as for susceptibility testing (see 2.5). Diluted yeast suspensions, 0.25 µg/ml of
styrylpyridinium compounds, and serially diluted fluconazole (0.0625–4 µg/ml) as well as 0.0625
µg/ml of fluconazole in combination with serially diluted styrylpyridinium compounds (0.25 to 16
µg/ml) were used for synergy evaluations (total volume of 100 μl, incubated without agitation at
37 °C for 24 h). After incubation, OD612 was read spectrophotometrically using the TECAN
GENios Pro™ plate reader. The sum of FICs (ΣFIC) was calculated using the equation ΣFIC =
FIC of agent A + FIC of agent B, where:
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MIC90 of compound A in combination
MIC90 of compound A alone
FIC (A) =
FIC (B) =
MIC90 of Fluconazole in combination
MIC90 of Fluconazole alone
MIC90 = concentration (µg/ml) required to inhibit the growth of cells by 90%. Synergy of
fluconazole and the compound was considered effective when the ΣFIC value was ≤0.5.
2.7. Electrochemical measurements
To study the interaction of the studied compounds with yeast, C. albicans cells were grown
with aeration in 10 ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 37 °C for
18 h. The overnight culture was diluted 1:50 with a fresh YPD medium and grown to A600 of 0.6 at
the same conditions. The cells were collected by centrifugation at 4 °C for 10 min at 3000g
(Heraeus Megafuge 16R, ThermoFisher Scientific, Germany). Pelleted cells were resuspended in a
modified RPMI-1640 medium, as described previously, to obtain 1/100 of the original cell culture
volume, kept on ice and used within 4 h.
Measurements of dissolved oxygen concentration in a medium were performed by adding
1.0 × 108 CFU yeast suspension to a thermostated (37 °C) and magnetically stirred reaction vessel,
containing 5 ml of RPMI-1640 medium (pH 7.0) and 32 µg/ml of the tested compound. At the end
of the experiment, solid Na2S2O4 was added to the vessel to approximately 20 mM to exhaust
dissolved oxygen. Oxygen concentration in the suspension after Na2S2O4 addition was set as 0%,
while the concentration in an RPMI-1640 medium before the addition of cells was set as 100%.
2.8. Cytotoxicity assay
Chinese hamster ovary (CHO-K1) cells were used for the evaluation of cytotoxicity of the
compounds. The cells were grown in a Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO,
Life Technologies), supplemented with fetal bovine serum (10% v/v, GIBCO, Life Technologies),
streptomycin/penicillin (2% v/v, GIBCO, Life Technologies), and amphotericin B (1% v/v;
GIBCO; Life Technologies) in 75-cm2 Falcon culture flasks under standard conditions of 5% CO2
in air at 37 °C, with culture medium renewal every 2–3 days. After reaching sufficient confluence,
the cells were passaged by trypsinization. The effects of the compounds on CHO-K1 cells were
examined using a 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
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(XTT) viability assay. This assay is based on the ability of metabolically active (live) cells to
reduce XTT tetrazolium salt, leading to the formation of orange-colored formazan compounds.
For the evaluation of cytotoxicity, CHO-K1 cells were grown in 96-well plates (starting
1 × 104 cells per well) for 24 h in a culture medium. After an incubation medium was carefully
aspirated (avoiding damage of the attached cells), wells were two times gently washed with PBS.
Solutions of the compounds (see 2.5) were added to each well and incubated for 24 h at 37 °C.
Controls consisted of the untreated cells. After an incubation media was removed from all wells,
100 µl of fresh DMEM and 50 µl of XTT solution were added to each well. The plates were
covered to avoid direct light and incubated for 2 h at 37 °C to induce the formation of a formazan
dye. After incubation, the plates were gently shaken, and absorbance at 492 nm was
spectrophotometrically read using the TECAN GENios Pro™ plate reader.
CHO-K1 cells grown under the same conditions without compounds served as a control.
Viability was calculated according to the equation:
absorbance of cells treated with compounds
Viability (%) =
× 100 %
absorbance of control cells
2.9. Statistical analysis
Statistical analysis was performed by using the GraphPad Prism 8 (ver. 8.01, 2018; GraphPad
Software Inc., San Diego, CA). All the experiments were performed in three biological replicates
with three technical repetitions. Results are expressed as means  standard error of the mean
(SEM).
3. Results and discussion
The synthesis of styrylpyridinium compounds Comps I-IV and VI-IX was described, and
fluorescence properties along with antimicrobial activities against bacteria were reported
elsewhere [11-13, 18-20]. It has been demonstrated that styrylpyridinium compounds (sometimes
described as pyridinium and quinolinium stilbenes) are more effective against Gram-positive than
Gram-negative bacteria [16]. This difference in sensitivity can be explained by the presence of the
outer membrane in Gram-negative bacteria [28].
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Due to the lack of information about the antifungal activity of the compounds, we evaluated
the activity of the known and newly synthesized styrylpyridinium derivatives against C. albicans
cells.
3.1. Characterization of styrylpyridinium compounds
We synthesized a set of compounds by changing two structural elements: substituents at the
phenyl ring of the styryl part and alkyl substituents at the pyridinium N-atom. In the first case, we
regulated an electronic structure of the conjugated system of the styrylpyridinium moiety.
In the second case, the length of pyridinium N-alkyl chain changed the lipophilicity of the
molecule and the processes of association with and incorporation into membranes. The alkyl-
chain varied from N-hexyl (Comp VII) followed by N-octyl (Comps I and VIII), N-decyl (Comp
II), N-dodecyl (Comps III, V, IX, and XI), N-octadecyl (Comps IV, VI, X, and XII) to N-icosyl
(Comp XIII)). In parallel, the logP values increased in a row (see Table 1): 0.59 (Comp VII) →
1.02 (Comp I), 1.60 (Comp VIII) → 2.03 (Comp II) → 3.04 (Comp III), 3.16 (Comp V), 4.37
(Comp IX), 3.27 (Comp XI) → 6.60 (Comp IV), 6.19 (Comp VI), 7.62 (Comp X), 8.29 (Comp
XII) → 8.86 (Comp XIII).
From the other point of view, in the first case, we used electron-donating or electron-
withdrawing substituents: powerful electron-withdrawing substituent 4′-CN, providing – I and –
M effects (Comp XI), medium electron-donating substituents 3′,4′-dimethoxy (Comps V and VI)
and 4′-methoxy (Comp IV), more powerful electron-donating substituents (providing – I and + M
effects) 4′-dimethylamino (Comps VII and VIII) and 4′-diethylamino (Comp IX), and fixed
dipropylamino moiety included in the julolidine framework (Comp X). For Comp IX, the
diethylamino group makes it less water soluble than the cyano group (Comp XI). Also, the
dipropylamino moiety in the tricyclic system makes Comp X less water soluble than one or two
methoxy groups (Comps IV and VI). Biphenyl substituents lead to low water solubility (Comps
XII and XIII).
In case of Comps I, II, and III, p-OH groups can be deprotonated. Calculated pKa values of
studied Comps I, II and III are at 8.87, near to published data at 8.7 – 8.5 [19]. In the cytosol,
hydroxy- and quinonoid-type derivatives have to be in equilibrium. Accordingly, we calculated
the coefficient of distribution for these compounds: logD at 6.30 – 7.41. It is quite close to logP
data, that is, the partition coefficient is not far from the distribution coefficient.
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All studied compounds have polar surface area less than 90 A2. It means that molecules can
permeate cell membranes, even blood-brain barrier.
3.2. Gene expression
In order to get more information about the clinical isolate SV1 from patients’ ascitic fluid, we
evaluated the gene expression of CDR1, CDR2, and MDR1 efflux pumps. We found that the
expression of CDR1 gene in SV1 cells was 5.59 ± 1.8-fold higher compared with ATCC10231
cells (Fig. 1). At the same time, the expression of CDR2 and MDR1 was a 2.80 ± 0.99-fold and a
12.86 ± 1.88-fold greater, respectively, in SV1 cells than ATCC10231 cells (Fig. 1).
3.3. Antifungal activity of styrylpyridinium compounds
For susceptibility evaluation, stock solutions of all tested compounds were prepared in
ethanol, serially diluted using an RPMI-1640 medium, and incubated with yeast suspensions in
96-well plates for 24 h as described above (chapter 2.5.).
In case of the compounds with a similar structure (Comps I, II, and III), fungicidal efficiency
was dependent on the length of N-alkyl chain. MIC50 and MIC90 of Comp II (C10H21) and Comp
III (C12H25) for ATCC10231 cells varied from 0.5 to 2 μg/ml, and for SV1 cells with increased
expression of efflux pump CDR1, CDR2, and MDR1 genes, higher concentrations ranging from 6
to 8 μg/ml were required. The pump mutant strains were more susceptible: concentrations between
0.25 and 1.5 μg/ml diminished cell growth up to 90%. At the same time, Comp I with the shortest
N-alkyl chain (C8H17) and the lowest logP (1.02) had higher MIC50 and MIC90 values for the cells
of almost all studied strains (Table 3).
Meanwhile, Comp V with a shorter N-alkyl chain (C12H25) compared to Comp VI with a
similar structure but longer N-alkyl chain (C18H37) showed a higher antifungal activity
considerably inhibiting yeast growth. MIC50 and MIC90 of Comp V for ATCC10231 cells were 0.5
and 1 μg/ml, respectively, and for mutant strains varied from 1 to 3 μg/ml. Concentrations up to 4
μg/ml were required to inhibit the growth of SV1 strain (Table 3). A similar effect of Comp V on
the cells of mutant strains, ATCC10231, and SV1 could indicate that this compound is not the
substrate of efflux pumps.
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Comps VII and VIII with short N-alkyl chains (C6H13 and C8H17) were also effective against
the cells of all C. albicans strains tested. Comp VII at the concentrations of 1 to 4 μg/ml inhibited
the growth of wt and efflux-defective strains by 50%, while concentrations at 16 μg/ml were
required to inhibit the growth of SV1 cells by 90% (Table 3). In case of Comp VIII with an octyl
chain, a stronger fungicidal effect was obtained along with the increase of logP. MIC50 for all
strains varied between 0.25 and 2 μg/ml, while MIC90 varied from 0.75 to 2 μg/ml for
ATCC10231 and efflux mutant strains, and higher concentrations were required for pumps
overexpressing SV1 cells (Table 3).
Comps IX and XI, having the same N-alkyl chain (C12H25), but different substituents at
position R2 (-NEt2 or -CN), demonstrated different antifungal effects. Comp IX had the lowest
MIC50 and MIC90 values among all compounds tested. ATCC10231 and SV1 strains demonstrated
the same susceptibility to this compound, MIC50 for the cells of both strains was 0.6 μg/ml, while
1 μg/ml was needed to inhibit the growth of these strains by 90%. At the same time, the efflux
pump mutants showed increased sensitivity to the tested compound, and 90% inhibition of the
yeast growth was observed at concentrations between 0.25 and 0.5 μg/ml. MIC50 of Comp XI for
wt and the mutant strains was in the range of 1 to 2.8 μg/ml, and MIC90 varied from 3 to 8 μg/ml.
The SV1 strain demonstrated increased resistance to this compound: more than 16 μg/ml was
required to inhibit the growth of these cells by 90% (Table 3).
Compounds having a medium long N-alkyl chains (C6H13 – C12H25) are the most appropriate
antifungal agents, we can use just this subtype of compounds to compare efficacy. Comp XI
having a strong electron withdrawing substituent CN has medium antifungal activity. Comp V
possessing methoxy group has quite high antifungal activity, but compounds possessing strong
electron donating groups (dimethylamino, diethylamino – Comps VII, VIII and IX) are the most
active (especially Comps VIII and IX; Comp VIII is preferable to Comp VII as the N-alkyl
chain C8H17 is preferable to C6H13).
Comps IV, VI, X, XII, and XIII with long alkyl chains (C18H37–C20H41) revealed high
hydrophobicity and low water solubility as well as their logP values were >6. According to the
Lipinski’s rule of 5, logP should be <5 for drug-likeness of a molecule. It coincides with obtained
experimental data: these compounds did not show any fungicidal activity at the concentrations of
<64 μg/ml. Due to this, these compounds were excluded from further analysis.
In case of fluconazole, MIC50 for the cells of ATCC10231 and mutant strains varied from
<0.06 to 0.5 μg/ml, whereas MIC90 for all strains was in the range of 0.06 to 1 μg/ml. Cells of the
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SV1 strain demonstrated the same sensitivity to fluconazole as ATCC10231 cells (Table 3).
Similar sensitivity of C. albicans cells to fluconazole has been reported by other 29-31]. Our
results showed that mdr1Δ strain had higher resistance to fluconazole compared with cdr1Δ,
cdr2Δ, and cdr1Δcdr2Δ strains. It is known [32] that Cdr1 pump in C. albicans has a higher
impact on fluconazole resistance than Cdr2 pump. This was confirmed by our results that cells
without Cdr1 pump showed lower MIC50/90 for fluconazole than cells bearing Cdr1 pump, but
lacking Cdr2 pump (Table 3). cdr1Δ and cdr1Δcdr2Δ strains showed a significant increase in
sensitivity to all styrylpyridinium compounds mentioned above, suggesting that these compounds
can be substrates of Cdr1 or Cdr2 pumps and competitive inhibitors of fluconazole efflux.
3.4. Antifungal activity of styrylpyridinium compounds in combination with fluconazole
Fluconazole is known as a first-choice drug to treat C. albicans infections. It is effective, well
tolerated, with predictable pharmacokinetics and can be used in different groups of patients, even
children [33]. However, C. albicans can develop resistance to fluconazole through various
mechanisms. Therefore, combined antifungal treatment is considered as a relevant tool. One of our
goals was to investigate a possible synergy between styrylpyridinium compounds and fluconazole
against C. albicans. For this purpose, the lowest non-active concentration of the compounds
against ATCC10231 and SV1 cells were tested in combination with various concentrations of
fluconazole.
Comps I, II, III, V, VIII, and XI in combination with fluconazole demonstrated the ΣFIC
values of 0.19–0.5, indicating significant synergism against ATCC10231, SV1, cdr1Δ, cdr2Δ, and
mdr1Δ cells (Table 4). A ΣFIC value of ≤ 0.5 was interpreted as synergy, whereas ΣFIC values
between 0.5 and 1.0 were interpreted as additive [34].
The most relevant synergism was achieved in case of SV1 strain, combining fluconazole with
Comp XI which resulted in ΣFIC of 0.19. Significant synergism between Comp IX and
fluconazole was obtained studying cells of ATCC10231, SV1 and cdr2Δ strains.
In cases when cdr1Δcdr2Δ strain cells demonstrated the highest sensitivity to all tested
compounds and fluconazole alone, no synergism of fluconazole in combination with
styrylpyridinium compounds was observed (Table 4). Moreover, fluconazole had no synergistic
effect with Comps VII and IX against cdr1Δ and mdr1Δ strains.
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There are many studies available on combinations of antifungal drugs with synthetic or natural
molecules. Namely, it has been reported that quercetin, a dietary flavonoid, in combination with
fluconazole acts synergistically against vulvovaginal C. albicans. Combination of these drugs has
ability to reduce adhesion and down-regulate expression of ALS1, ALS3, HWP1, SUN41, UME6,
and ECE1 genes responsible for biofilm formation [35]. Pure polyphenol curcumin I with
fluconazole or amphotericin B synergistically triggers the generation of reactive oxygen species
and induces apoptosis, which could be reversed by antioxidant ascorbic acid [36]. The advantages
of combined treatment are the increased activities of both compounds resulting in a reduced
number of resistant microorganisms and the lower toxicity of both compounds [37].
3.5. Effects of styrylpyridinium compounds on C. albicans respiration
In order to know how fast the compounds enter C. albicans cells and interact with
mitochondria, we monitored the induced changes in cell respiration. For this, we measured the
concentration of dissolved oxygen in an RPMI-1640 medium in the presence of styrylpyridinium
compounds (Fig. 2).
In the absence of compounds in the medium, the added cells immediately started intensive
respiration, and after 6 min, only 25% of the initial dissolved oxygen concentration was left in the
medium. Similar intensity of respiration was obtained after the addition of fluconazole (Fig. 2). In
the presence of Comps I and XI, the initial rate of respiration was lower, and during the first 6
min, the concentration of dissolved oxygen decreased to 50% or 40%, respectively (Fig. 2).
In the presence of other compounds, lower stable dissolved oxygen concentrations in the
medium were achieved. At equilibrium state, the dissolved oxygen concentration varied from 40%
(Comp II) to 70% (Comp III). In the presence of Comp IX, the rate of C. albicans respiration
gradually decreased and the dissolved oxygen concentration was approaching to the initial one. At
the end of experiments, solid Na2S2O4 was added (~20 mM), and the concentration of dissolved
oxygen dropped to 0 (Fig. 2).
Summarizing the results on the effects of the compounds on SV1 cell respiration, we can
conclude that Comp IX strongly inhibited respiration, while Comp XI had the smallest effect on
this process. Other compounds only slowed down respiration to a different extent.
3.6. Viability of CHO-K1 cells after treatment with styrylpyridinium compounds
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Fungi share similar cellular and biochemical characteristics with other eukaryotic cells, and
therefore, fungicidal agents may adversely affect mammalian cells [38]. However, it was
demonstrated that usage of sodium nitroprusside or salicylhydroxamic acid [39] resulted in the
efficiently inhibited respiration of C. albicans without causing damage to mammalian cells.
Consequently, we evaluated the cytotoxicity of the synthesized styrylpyridinium compounds. To
explore cytotoxicity, a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT)-based assay was applied, which shows the ability of metabolically active (live) cells to
reduce tetrazolium salt of XTT, leading to the formation of orange formazan compounds.
Comp XI had the weakest effect on the viability of CHO-K1 cells. After treatment with MIC90
(16 µg/ml), which was required to inhibit the growth of SV1 cells, a 30% decrease in the viability
of CHO-K1 cells was recorded (Fig. 3).
Comp IX possessed the highest toxicity: at MIC50 and MIC90 concentrations diminished
viability by 70%, and a 100% inhibition of CHO-K1 cell growth was registered at 2 μg/ml
concentration of this compound (Fig. 3A). In addition, Comp III at fungicidal concentrations (up
to 8 μg/ml) affected CHO-K1 cells by decreasing viability to 10%. Comps I, II, V, VII, and VIII
at MIC50 concentrations reduced CHO-K1 cell viability to 30%–40%, and MIC90 concentrations of
Comps I and VIII decreased the viability of mammalian cells to 10% and 0%, respectively.
Comps II, V, and VII at MIC90 concentrations reduced viability to 35%–55%, respectively (Fig.
3A). Styrylpyridinium compounds in combination with fluconazole had no considerable effect on
the growth of CHO-K1 cells compared with the effect of compounds on the cell viability when
tested separately (Fig. 3B).
In summary, the evaluation of cytotoxicity showed the same tendency as respiration
measurements: Comp IX was the most toxic, and Comp XI at MIC90 concentration (16 μg/ml)
demonstrated the lowest effect on the viability of CHO-K1 cells.
4. Conclusions
The present study demonstrated that newly and previously synthesized styrylpyridinium
compounds (Comp I-III, V, VII-IX, and XI) with N-alkyl chains not longer than C12H25 are
effective against all tested C. albicans cells. Their logP values were <5, in accordance with the
Lipinski’s rule of 5. Compounds possessing logP more than 6 (according the Molinspiration
method) or 9 (according to PerkinElmer Information method) do not have antifungal activity. At
fungicidal concentrations, Comp XI with the CN- group as a substituent was the least toxic to
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mammalian cells, showed the most effective synergism with fluconazole, and only slightly
inhibited the respiration of C. albicans. This compound fungicidally acted against all tested yeast
strains, but the highest concentration was required to inhibit the growth of SV1 cells with
increased CDR1, CDR2, and MDR1 gene expression. These results suggest that Comp XI could
be a substrate of efflux pumps. Comp IX exhibited the strongest fungicidal properties and
effectively blocked the respiration of SV1 cells. However, the toxicity of this compound to
mammalian cells was also rather high. Studied compounds have polar surface area less than 90 A2
and such molecules can permeate membranes.
Summarizing these findings, delocalization of the positive charge of the pyridinium moiety of
Comp IX, in conjugation with strong electron-donating diethylamino group at the phenyl group
(push-pull mechanism), results in more even distribution of electrons and diminution of polarity.
Comp XI with a strong electron-withdrawing CN group does not promote interaction with the
cationic N atom of pyridinium. The results of our study indicate that styrylpyridinium could be a
promising candidate in the development of new antifungal drugs. Therefore, our next step will be
a comprehensive evaluation of styrylpyridinium compounds acting as inhibitors of efflux pumps.
Funding
This research was funded by the European Social Fund under “The Development of
Competences of Scientists, other Researchers and Students through Practical Research Activities”
measure (No. 09.3.-LMT-K-712-02-0200) under grant agreement with the Research Council of
Lithuania (LMTLT). Support of the Latvian group project NFI/R/2014/051 is acknowledged.
Acknowledgments
We wish to thank Prof. Dominique Sanglard (Institute of Microbiology, University of
Lausanne and University Hospital Center, Lausanne, Switzerland) for kindly providing Candida
albicans mutant strains, and Laboratory of Microbiology (Republican Panevezys Hospital,
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Lithuania) for providing clinical isolate strain SV1. Also, we thank Dr. Z. Kalme and I. Bruvere
for the performance of some steps of synthesis.
Author contributions
SV designed and performed biological experiments of this work and prepared the manuscript. NK
and SS participated at the susceptibility evaluation of compounds on Candida cells. LB was
responsible for the outset of the chemical part. LK was responsible for the synthesis of a major
part of compounds. BV was responsible for compounds analysis, structure determination, and the
final chemical part of the project. GD was the leader of the chemical part of the project,
responsible for the analysis of the structure-activity relationship of compounds. RD participated in
designing of the experiments, revised the manuscript, leader of the team. All authors have read
and approved the final manuscript.
Competing financial interests
The authors declare no competing financial interests.
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Figure legends
Scheme 1. General methods for the synthesis of N-alkyl styrylpyridinium dyes I–XIII.
Reagents and conditions: (a) 4-methylpyridine, appropriate alkyl bromide or tosylate, MeCN,
24 h, 80 °C; (b) N-alkyl pyridinium bromide or tosylate, substituted benzaldehyde, MeOH,
piperidine, reflux, 5 h.
Fig. 1. CDR1, CDR2, and MDR1 gene expression in SV1 cells.
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Fig. 2. Intensity of respiration of C. albicans SV1 cells. The concentration of dissolved oxygen
was measured as described in Materials and methods. Concentrated yeast suspension was added to
the vessel, containing 5 mL of RPMI-1640 medium (pH 7.0) and 32 µg/ml of the tested
compound, to the final concentration of 1.0 × 108 cells/ml. Solid Na2S2O4 was added to the vessel
(~20 mM concentration).
Fig. 3. Effect of styrylpyridinium compounds and fluconazole on viability of CHO-K1 cells.
CHO-K1 cells were grown 24 h in 96-well plates filled with DMEM medium in the presence of
various concentrations of styrylpyridinium compounds (A), or serially diluted fluconazole (FLC)
in combination with a constant concentration (0.25 μg/ml) of styrylpyridinium compounds (B).
Viability of cells in the absence of tested compounds corresponds to 100%. The tested compounds
were prepared as described above (chapter 2.5), were added to each well containing the attached
cells, and were incubated at 37 °C for 24 h.
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Table 1. Styrylpyridinium compounds used in this study
Compounds R1
R2
R3
R
X–
logP /logP’
PSA, A2
Reference
Comp I
H
OH
H
C8H17
Br–
1.02/6.32
*8.87
23.24
[12]
#7.4
†6.30
Comp II
H
OH
H
C10H21
Br–
2.03/7.42
*8.87
23.24
[19]
#7.4
†7.41
Comp III
Comp IV
Comp V
H
OH
H
C12H25
C18H37
C12H25
C18H37
C6H13
Br–
Br–
3.04/7.25
6.60/10.24
3.16/7.21
6.19/9.88
0.59/5.85
1.60/6.95
4.37/8.74
7.62/10.96
3.27/7.39
8.29/12.28
8.86/13.17
23.24
12.24
21.47
21.47
6.25
[20]
[21]
H
OMe
OMe
OMe
NMe2
NMe2
NEt2
N
H
OMe
OMe
H
H
Br–
this study
[22]
Comp VI
Comp VII
Comp VIII
Comp IX
Comp X
H
Br–
–
H
MeC6H4SO3
Br–
Br–
[13]
H
H
C8H17
6.25
[11]
H
H
C12H25
C18H37
C12H25
C18H37
C20H41
6.25
this study
this study
this study
this study
this study
(CH2)3
H
(CH2)3
H
Br–
Br–
6.25
Comp XI
Comp XII
Comp XIII
CN
26.8
H
Ph
H
Br–
3.01
H
Ph
H
Br–
3.01
*pKa; #logD; †acid
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Table 2. C. albicans efflux pump mutant strains used in this study
Strain
Genotype
Reference
[25]
DSY448
DSY653
DSY465
DSY654
cdr1Δ::hisG-URA3 hisG/cdr1Δ::hisG
cdr2Δ::hisG-URA3-hisG/cdr2Δ::hisG
mdr1Δ::hisG-URA3-hisG/ mdr1Δ::hisG
cdr1Δ::hisG/cdr1Δ::hisG cdr2Δ::hisG-
URA3-hisG/cdr2Δ::hisG
[26]
[25]
[26]
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Table 3. In vitro activity of styrylpyridinium compounds against C. albicans
Strains
Compound
SV1
R1-R3
R
logP
cdr1Δ
cdr2Δ
mdr1Δ
MIC50
0.25
2
cdr1Δcdr2Δ
ATCC10231
MIC50 MIC90
MIC50 MIC90 MIC50
MIC90
0.12
4
MIC50 MIC90
MIC90 MIC50
MIC90
Fluconazole
Comp I
0.15
1
0.5
4
0.15
4
0.5
>16
8
<0.06
0.5
0.25
0.5
ND
1
0.12
1
0.5
5
1
<0.06
0.5
0.5
0.75
ND
1
0.06
4
-
-
-
6
4-OH
8
1.02
2.03
3.04
6.60
3.16
6.19
0.59
1.60
4.37
7.62
3.27
8.29
8.86
Comp II
0.5
1.5
>64
0.5
>64
4
2
6
1.5
1.5
ND
2
0.5
0.5
ND
1
2
0.75
0.75
ND
1
3
1.5
1.4
ND
1.5
ND
1.5
0.75
0.25
ND
3
4-OH
10
12
18
12
18
6
Comp III
Comp IV
Comp V
2
7
8
1.6
ND
3
1.5
ND
2
4-OH
>64
1
>64
3
>64
4
4-OMe
3,4-(OMe)2
3,4-(OMe)2
4-NMe2
4-NMe2
4-NEt2
Me3NMe3
4-CN
Comp VI
Comp VII
Comp VIII
Comp IX
Comp X
>64
8
>64
4
>64
16
ND
1
ND
1.8
0.9
0.5
ND
4
ND
1.5
0.5
0.4
ND
2
ND
8
ND
1
ND
2
ND
1
0.5
0.6
>64
2.8
>64
>64
2
2
8
0.4
0.37
ND
1
4
0.4
0.8
0.25
ND
6
0.25
<0.25
ND
1
8
1
0.6
>64
4
1
0.5
ND
4
<0.25
ND
1
12
18
12
18
20
>64
8
>64
>16
>64
>64
Comp XI
Comp XII
Comp XIII
>64
>64
>64
>64
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4-Ph
4-Ph
MIC50 = concentration (µg/ml) required to inhibit the growth of cells by 50%, MIC90 = concentration (µg/ml) required to inhibit the growth of
cells by 90%, ND = not determined;
R - number of C atoms in N-alkyl chain.
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Table 4. Synergistic effects of styrylpyridinium compounds and fluconazole against
C. albicans
Strains
SV1
cdr1Δ
cdr2Δ
mdr1Δ
cdr1Δcdr2Δ
ATCC10231
Comp I
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
0.5
0.5
0.38
0.31
0.31
0.38
0.38
0.28
0.38
0.19
0.5
0.45
0.38
0.4
0.45
0.41
0.42
0.25
>0.5
0.38
>0.5
0.4
Comp II
Comp III
Comp V
Comp VII
Comp VIII
Comp IX
Comp XI
0.41
0.41
0.31
>0.5
0.32
>0.5
0.27
0.38
0.5
0.29
0.5
0.31
0.38
0.38
0.38
0.38
0.38
0.5
The results are presented as ΣFIC for the tested styrylpyridinium compounds, and the values
in bold indicate significant synergism.
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